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Several marine drugs exert anticancer effects by inducing oxidative stress, which becomes overloaded and kills cancer cells when redox homeostasis is imbalanced. The downregulation of antioxidant signaling induces oxidative stress, while its upregulation attenuates oxidative stress. Marine drugs have miRNA-modulating effects against cancer cells. However, the potential antioxidant targets of such drugs have been rarely explored. This review aims to categorize the marine-drug-modulated miRNAs that downregulate their antioxidant targets, causing oxidative stress in anticancer treatments. We also categorize the downregulation of oxidative-stress-inducing miRNAs in antioxidant protection among non-cancer cells. We summarize the putative antioxidant targets of miRNA-modulating marine drugs by introducing a bioinformatics tool (miRDB). Finally, the marine drugs affecting antioxidant targets are surveyed. In this way, the connections between marine drugs and their modulating miRNA and antioxidant targets are innovatively categorized to provide a precise network for exploring their potential anticancer functions and protective effects on non-cancer cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Manoalide provides preferential antiproliferation of oral cancer but is non-cytotoxic to normal cells by modulating reactive oxygen species (ROS) and apoptosis. Although ROS interplays with endoplasmic reticulum (ER) stress and apoptosis, the influence of ER stress on manoalide-triggered apoptosis has not been reported. The role of ER stress in manoalide-induced preferential antiproliferation and apoptosis was assessed in this study. Manoalide induces a higher ER expansion and aggresome accumulation of oral cancer than normal cells. Generally, manoalide differentially influences higher mRNA and protein expressions of ER-stress-associated genes ( PERK , IRE1α , ATF6 , and BIP ) in oral cancer cells than in normal cells. Subsequently, the contribution of ER stress on manoalide-treated oral cancer cells was further examined. ER stress inducer, thapsigargin, enhances the manoalide-induced antiproliferation, caspase 3/7 activation, and autophagy of oral cancer cells rather than normal cells. Moreover, N -acetylcysteine, an ROS inhibitor, reverses the responses of ER stress, aggresome formation, and the antiproliferation of oral cancer cells. Consequently, the preferential ER stress of manoalide-treated oral cancer cells is crucial for its antiproliferative effect.

Noncoding RNAs (ncRNAs) regulation of various diseases including cancer has been extensively studied. Reactive oxidative species (ROS) elevated by oxidative stress are associated with cancer progression and drug resistance, while autophagy serves as an ROS scavenger in cancer cells. However, the regulatory effects of ncRNAs on autophagy and ROS in various cancer cells remains complex. Here, we explore how currently investigated ncRNAs, mainly miRNAs and lncRNAs, are involved in ROS production through modulating antioxidant genes. The regulatory effects of miRNAs and lncRNAs on autophagy-related (ATG) proteins to control autophagy activity in cancer cells are discussed. Moreover, differential expression of ncRNAs in tumor and normal tissues of cancer patients are further analyzed using The Cancer Genome Atlas (TCGA) database. This review hypothesizes links between ATG genes- or antioxidant genes-modulated ncRNAs and ROS production, which might result in tumorigenesis, malignancy, and cancer recurrence. A better understanding of the regulation of ROS and autophagy by ncRNAs might advance the use of ncRNAs as diagnostic and prognostic markers as well as therapeutic targets in cancer therapy., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The sensitizing effect of chromone-derived compounds on UVC-induced proliferation inhibition has not been comprehensively investigated so far. The subject of this study was to examine the proliferation change of oral cancer cells while using the combined treatment of UVC (254 nm) with our previously developed sulfonyl chromen-4-ones (CHW09), namely UVC/CHW09. Cell viability, apoptosis, oxidative stress, and DNA damage for the individual and combined treatments for UVC and/or CHW09 were examined in oral cancer Ca9-22 cells. In 24 h MTS assay, UVC (30 J/m 2 ; UVC30), or CHW09 (25 and 50 µg/mL; namely, CHW09-25 and CHW09-50) show 54%, 59%, and 45% viability. The combined treatment (UVC30/CHW09-25 and UVC30/CHW09-50) show lower cell viability (45% and 35%). Mechanistically, UVC/CHW09 induced higher apoptosis than individual treatments and untreated control, which were supported by the evidence of flow cytometry for subG1, annexin V/7-aminoactinomycin D, pancaspase and caspases 3/7 activity, and western blotting for cleaved poly(ADP-ribose) polymerase. Moreover, this cleaved PARP expression was downregulated by pancaspase inhibitor Z-VAD-FMK. UVC/CHW09 showed higher oxidative stress than individual treatments and untreated control in terms of flow cytometry for reactive oxygen species, mitochondrial membrane potential, and mitochondrial mass. Furthermore, UVC/CHW09 showed higher DNA damage than individual treatments and untreated control in terms of flow cytometry for H2A histone family member X and 8-oxo-2'-deoxyguanosine. In conclusion, combined treatment UVC/CHW09 suppresses proliferation, and promotes apoptosis, oxidative stress, and DNA damage against oral cancer cells, providing a novel application of sulfonyl chromen-4-ones in order to sensitize UVC induced proliferation inhibition for oral cancer therapy.

Withaferin A (WFA) has been reported to inhibit cancer cell proliferation based on high cytotoxic concentrations. However, the low cytotoxic effect of WFA in regulating cancer cell migration is rarely investigated. The purpose of this study is to investigate the changes in migration and mechanisms of oral cancer Ca9-22 cells after low concentrations of WFA treatment. WFA under 0.5 μM at 24 h treatment shows no cytotoxicity to oral cancer Ca9-22 cells (~95% viability). Under this condition, WFA triggers reactive oxygen species (ROS) production and inhibits 2D (wound healing) and 3D cell migration (transwell) and Matrigel invasion. Mechanically, WFA inhibits matrix metalloproteinase (MMP)-2 and MMP-9 activities but induces mRNA expression for a group of antioxidant genes, such as nuclear factor, erythroid 2-like 2 ( NFE2L2 ), heme oxygenase 1 ( HMOX1 ), glutathione-disulfide reductase ( GSR ), and NAD(P)H quinone dehydrogenase 1 ( NQO1 )) in Ca9-22 cells. Moreover, WFA induces mild phosphorylation of the mitogen-activated protein kinase (MAPK) family, including extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 expression. All WFA-induced changes were suppressed by the presence of ROS scavenger N -acetylcysteine (NAC). Therefore, these results suggest that low concentration of WFA retains potent ROS-mediated anti-migration and -invasion abilities for oral cancer cells.

To achieve preferential effects against cancer cells but less damage to normal cells is one of the main challenges of cancer research. In this review, we explore the roles and relationships of oxidative stress-mediated apoptosis, DNA damage, ER stress, autophagy, metabolism, and migration of ROS-modulating anticancer drugs. Understanding preferential anticancer effects in more detail will improve chemotherapeutic approaches that are based on ROS-modulating drugs in cancer treatments., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Nepenthes plants are regarded as a kind of Traditional Chinese Medicine for several diseases but its anticancer activity remain unclear. The subject of this study is to evaluate the antiproliferation effects on oral cancer cells by Nepenthes plants using ethyl acetate extract of Nepenthes adrianii x clipeata (EANA). Cell viability was detected using MTS assay. Its detailed mechanisms including cell cycle, apoptosis, oxidative stress, and DNA damage were explored by flow cytometry or western blotting. For 24 hours EANA treatment, five kinds of oral cancer cells (CAL 27, Ca9-22, OECM-1, HSC-3, and SCC9) show IC 50 values of cell viability ranging from 8 to 17 μg/mL but the viability of normal oral cells (HGF-1) remains over 80%. Subsequently, CAL 27 and Ca9-22 cells with high sensitivity to EANA were chosen to investigate the detailed mechanism. EANA displays the time course and concentration effects for inducing apoptosis based on flow cytometry (subG1 and annexin V analyses) and western blotting [cleaved poly (ADP-ribose) polymerase (c-PARP)]. Oxidative stress and DNA damage were induced by EANA treatments in oral cancer cells through reactive oxygen species (ROS), mitochondrial membrane potential disruption, mitochondrial superoxide, and γH2AX. All these changes of EANA treatments in oral cancer cells were reverted by the ROS scavenger N-acetylcysteine pretreatment. Therefore, EANA induces preferential killing, apoptosis, and DNA damage against oral cancer cells through oxidative stress., (© 2019 Wiley Periodicals, Inc.)

Several functionalized chromones, the key components of naturally occurring oxygenated heterocycles, have anticancer effects but their sulfone compounds are rarely investigated. In this study, we installed a sulfonyl substituent to chromen-4-one skeleton and synthesized CHW09 to evaluate its antioral cancer effect in terms of cell viability, cell cycle, apoptosis, oxidative stress, and DNA damage. In cell viability assay, CHW09 preferentially kills two oral cancer cells (Ca9-22 and CAL 27), less affecting normal oral cells (HGF-1). Although CHW09 does not change the cell cycle distribution significantly, CHW09 induces apoptosis validated by flow cytometry for annexin V and by western blotting for cleaved poly(ADP-ribose) polymerase (PARP), and caspases 3/8/9. These apoptosis signaling expressions are partly decreased by apoptosis inhibitor (Z-VAD-FMK) or free radical scavenger (N-acetylcysteine). Furthermore, CHW09 induces oxidative stress validated by flow cytometry for the generations of reactive oxygen species (ROS) and mitochondrial superoxide (MitoSOX), and the suppression of mitochondrial membrane potential (MMP). CHW09 also induces DNA damage validated by flow cytometry for the increases of DNA double strand break marker γH2AX and oxidative DNA damage marker 8-oxo-2'-deoxyguanosine (8-oxodG). Therefore, our newly synthesized CHW09 induces apoptosis, oxidative stress, and DNA damage, which may lead to preferential killing of oral cancer cells compared with normal oral cells., (© 2018 Wiley Periodicals, Inc.)

Objective: Sinuleptolide, a soft corals-derived bioactive norditerpenoid, is a marine natural product with a potent anti-inflammatory effect. We evaluate the potential anti-oral cancer effects of sinuleptolide and investigate the possible mechanisms involved., Designs: Cell viability, cell cycle, apoptosis, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and DNA damage analyses were performed., Results: In a cell viability assay, we found that sinuleptolide is dose-responsively antiproliferative against oral gingival cancer Ca9-22 cells but less harmful to normal human gingival fibroblast (HGF-1) cells (P<0.001). In cell cycle analysis, sinuleptolide induced subG1 accumulation at a higher dose and led to G2/M arrest of Ca9-22 cells (P<0.005). Apoptosis was significantly increased in sinuleptolide-treated Ca9-22 cells based on annexin V and poly(ADP-ribose) polymerase (PARP) expressions (P<0.05-0.0001). Based on flow cytometer analysis, sinuleptolide also induced the generation of ROS and decreased MMP in a dose-responsive manner (P<0.05-0.0001). DNA damage increased dose-responsively after sinuleptolide treatments (P < 0.001) based on comet and γH2AX assays., Conclusion: Sinuleptolide can induce an antiproliferation of oral cancer Ca9-22 cells involving apoptosis, oxidative stress and DNA damage, suggesting that sinuleptolide represents a potential chemotherapeutic drug for oral cancer treatment., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

We have previously found that the aqueous extract of Gracilaria tenuistipitata (AEGT) and its partitioned fractions had antioxidant properties in biochemical assays. Although the butanol-partitioned fraction of AEGT (AEGT-pBuOH) had a stronger antioxidant performance than AEGT, its biological effects are still unknown. In this study, the cellular responses of oral cancer cells to AEGT-pBuOH were monitored in terms of cell viability, cell cycle progression, apoptosis, and oxidative stress responses. In an ATP content assay, the cell viability of oral cancer cells treated with AEGT-pBuOH was dose responsively inhibited (p < 0.005). For flow cytometry, AEGT-pBuOH was also found to dose responsively induce cell cycle disturbance by propidium iodide (PI) staining and to induce apoptosis by annexin V/PI and pan-caspase staining (p < 0.005). In AEGT-pBuOH-treated oral cancer cells, the reactive oxygen species (ROS) was increased and mitochondrial membrane potential was decreased in a dose-response manner (p < 0.005). These results suggest that AEGT-pBuOH inhibited the proliferation and induced apoptosis of oral cancer cells involving the ROS generation and mitochondrial depolarization.

Roe protein hydrolysates were reported to have antioxidant property but the anticancer effects were less addressed, especially for oral cancer. In this study, we firstly used the ultrafiltrated roe hydrolysates (URH) derived from giant grouper (Epinephelus lanceolatus) to evaluate the impact of URH on proliferation against oral cancer cells. We found that URH dose-responsively reduced cell viability of two oral cancer cells (Ca9-22 and CAL 27) in terms of ATP assay. Using flow cytometry, URH-induced apoptosis of Ca9-22 cells was validated by morphological features of apoptosis, sub-G1 accumulation, and annexin V staining in dose-responsive manners. URH also induced oxidative stress in Ca9-22 cells in terms of reactive oxygen species (ROS)/superoxide generations and mitochondrial depolarization. Taken together, these data suggest that URH is a potential natural product for antioral cancer therapy.

Background: Grape seeds extract (GSE) is a famous health food supplement for its antioxidant property. Different concentrations of GSE may have different impacts on cellular oxidative/reduction homeostasis. Antiproliferative effect of GSE has been reported in many cancers but rarely in oral cancer., Methods: The aim of this study is to examine the antioral cancer effects of different concentrations of GSE in terms of cell viability, apoptosis, reactive oxygen species (ROS), mitochondrial function, and DNA damage., Results: High concentrations (50-400 μg/ml) of GSE dose-responsively inhibited proliferation of oral cancer Ca9-22 cells but low concentrations (1-10 μg/ml) of GSE showed a mild effect in a MTS assay. For apoptosis analyses, subG1 population and annexin V intensity in high concentrations of GSE-treated Ca9-22 cells was increased but less so at low concentrations. ROS generation and mitochondrial depolarization increased dose-responsively at high concentrations but showed minor changes at low concentrations of GSE in Ca9-22 cells. Additionally, high concentrations of GSE dose-responsively induced more γH2AX-based DNA damage than low concentrations., Conclusions: Differential concentrations of GSE may have a differentially antiproliferative function against oral cancer cells via differential apoptosis, oxidative stress and DNA damage.

Indoxyl sulfate (IS) contributes to oxidative stress and endothelial dysfunction in chronic kidney disease patients. However, the role of mitochondria in IS-induced oxidative stress is not very clear. In this study, we examined whether mitochondria play a pivotal role in modulating the effects of antioxidants during IS treatment. In the context of human umbilical vein endothelial cells, we found that IS had a dose-dependent antiproliferative effect. In addition, we used flow cytometry to demonstrate that the level of reactive oxygen species increased in a dose-dependent manner after treatment with IS. High doses of IS also corresponded to increased mitochondrial depolarization and decreased mitochondrial DNA copy number and mitochondrial mass. However, these effects could be reversed by the addition of antioxidants, namely, vitamin C and N-acetylcysteine. Thus, our results suggest that IS-induced oxidative stress and antiproliferative effect can be attributed to mitochondrial dysfunction and impaired biogenesis and that these processes can be protected by treatment with antioxidants.

Data obtained through high-throughput technologies have gradually revealed that a unique stratified epithelial architecture of human skin along with the antioxidant-response pathways provided vital defensive mechanisms against UV radiation. However, it is noteworthy that skin is a major target for toxic insult by UV radiations that can alter its structure and function. Substantial fraction of information has been added into the existing pool of knowledge related to natural products mediated biological effects in UV exposed skin cells. Accumulating evidence has started to shed light on the potential of these bioactive ingredients as protective natural products in cosmetics against UV photodamage by exerting biological effects mainly through wide ranging intracellular signalling cascades of oxidative stress and modulation of miRNAs. In this review, we have summarized recently emerging scientific evidences addressing underlying mechanisms of UV induced oxidative stress and deregulation of signalling cascades and how natural products can be used tactfully to protect against UV induced harmful effects.

Many red algae-derived natural products are known to have anticancer effects. The biological functions of the red alga Solieria robusta from the Karachi coast (Pakistan) remain unclear. Here, we prepared a methanolic extracts of S. robusta (MESR) to examine its possible anti-oral cancer effects and the corresponding mechanism of action. Cell viability of MESR-incubated oral cancer Ca9-22 cells was dose-responsively decreased (p<0.001). According to a propidium iodide (PI)-based assay the cell cycle distribution was dramatically changed, especially for subG1 accumulation. Annexin V/PI assay of apoptosis using flow cytometry also showed that MESR-incubated Ca9-22 cells were dose-responsively increased (p<0.001). For evaluation of oxidative stress in MESR-incubated Ca9-22 cells, we found that reactive oxygen species (ROS) were overexpressed dose- and time-responsively and mitochondrial depolarization was also increased (p<0.001). Taken together, MESR showed inhibitory effects on oral cancer proliferation coupled with apoptosis and oxidative stress.

Background: Methanolic extracts of Gracilaria tenuistipitata (MEGT) were obtained from the edible red algae. Previously, we found that water extract of G. tenuistipitata was able to modulate oxidative stress-induced DNA damage and its related cellular responses., Methods: In this study, the methanol extraction product MEGT was used to evaluate the cell growth inhibition in oral cancer cells and its possible mechanism was investigated., Results: The cell viability of MEGT treated Ca9-22 oral cancer cell line was significantly decreased in a dose-response manner (p < 0.05). The sub-G1 population and annexin V intensity of MEGT-treated Ca9-22 cancer cells were significantly increased in a dose-response manner (p < 0.0005 and p < 0.001, respectively). The γH2AX intensities of MEGT-treated Ca9-22 cancer cells were significantly increased in a dose-response manner (p < 0.05). The reactive oxygen species (ROS) and glutathione (GSH)-positive intensities of MEGT-treated Ca9-22 oral cancer cells were significantly increased and decreased, respectively, in a dose-response manner (p < 0.05). The DiOC2(3) intensity for mitochondrial membrane potential (MMP) of MEGT-treated Ca9-22 cancer cells was significantly decreased in a dose-response manner (p < 0.05)., Conclusions: These results indicated that MEGT had apoptosis-based cytotoxicity against oral cancer cells through the DNA damage, ROS induction, and mitochondrial depolarization. Therefore, MEGT derived from the edible algae may have potential therapeutic effects against oral squamous cell carcinoma (OSCC).

The expression of proliferating cell nuclear antigen (PCNA) promoter was moderately induced in UV-irradiated, quiescent human and rodent cells. The induction was independent of tumor suppressor gene p53, because the PCNA expression was UV-inducible in the subclones of human fibroblasts in which the activity of p53 was abrogated by human papilloma virus E6. Furthermore, the induction did not depend on DNA repair, since PCNA was UV inducible in UVL-10 and xrs-6 cells, in which nucleotide excision repair and double-stranded repair, respectively, are largely compromised. However, the induction was inhibited by antioxidant N-acetylcysteine. The role of oxidative stress observed here is consistent with the previous finding that the proximal AP-1 site is critical to the UV inducibility of PCNA promoter.

The anti-oral cancer effects of santamarine (SAMA), a Michelia compressa var. compressa-derived natural product, remain unclear. This study investigates the anticancer effects and acting mechanism of SAMA against oral cancer (OC-2 and HSC-3) in parallel with normal (Smulow–Glickman; S-G) cells. SAMA selectively inhibits oral cancer cell viability more than normal cells, reverted by the oxidative stress remover N-acetylcysteine (NAC). The evidence of oxidative stress generation, such as the induction of reactive oxygen species (ROS) and mitochondrial superoxide and the depletion of mitochondrial membrane potential and glutathione, further supports this ROS-dependent selective antiproliferation. SAMA arrests oral cancer cells at the G2/M phase. SAMA triggers apoptosis (annexin V) in oral cancer cells and activates caspases 3, 8, and 9. SAMA enhances two types of DNA damage in oral cancer cells, such as γH2AX and 8-hydroxy-2-deoxyguanosine. Moreover, all of these anticancer mechanisms of SAMA are more highly expressed in oral cancer cells than in normal cells in concentration and time course experiments. These above changes are attenuated by NAC, suggesting that SAMA exerts mechanisms of selective antiproliferation that depend on oxidative stress while maintaining minimal cytotoxicity to normal cells.

This in vitro study examines the anti-oral cancer effects and mechanisms of a combined X-ray/SK2 treatment, i.e., X-ray and 6-n-butoxy-10-nitro-12,13-dioxa-11-azatricyclo[7.3.1.02,7]trideca-2,4,6,10-tetraene (SK2). ATP cell viability and flow cytometry-based cell cycle, apoptosis, oxidative stress, and DNA damage assessments were conducted. The X-ray/SK2 treatment exhibited lower viability in oral cancer (Ca9-22 and CAL 27) cells than in normal (Smulow–Glickman, S-G) cells, i.e., 32.0%, 46.1% vs. 59.0%, which showed more antiproliferative changes than with X-ray or SK2 treatment. Oral cancer cells under X-ray/SK2 treatment showed slight subG1 and G2/M increments and induced high annexin V-monitored apoptosis compared to X-ray or SK2 treatment. The X-ray/SK2 treatment showed higher caspase 3 and 8 levels for oral cancer cells than other treatments. X-ray/SK2 showed a higher caspase 9 level in CAL 27 cells than other treatments, while Ca9-22 cells showed similar levels under X-ray and/or SK2. The X-ray/SK2 treatment showed higher reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) depletion than other treatments. Meanwhile, the mitochondrial superoxide (MitoSOX) and glutathione levels in X-ray/SK2 treatment did not exhibit the highest rank compared to others. Moreover, oral cancer cells had higher γH2AX and/or 8-hydroxy-2-deoxyguanosine levels from X-ray/SK2 treatment than others. All these measurements for X-ray/SK2 in oral cancer cells were higher than in normal cells and attenuated by N-acetylcysteine. In conclusion, X-ray/SK2 treatment showed ROS-dependent enhanced antiproliferative, apoptotic, and DNA damage effects in oral cancer cells with a lower cytotoxic influence on normal cells.

Triple-negative breast cancer (TNBC) is insensitive to target therapy for non-TNBC and needs novel drug discovery. Extracts of the traditional herb Boesenbergia plant in Southern Asia exhibit anticancer effects and contain novel bioactive compounds but merely show cytotoxicity. We recently isolated a new compound from B. stenophylla, stenophyllol B (StenB), but the impact and mechanism of its proliferation-modulating function on TNBC cells remain uninvestigated. This study aimed to assess the antiproliferative responses of StenB in TNBC cells and examine the drug safety in normal cells. StenB effectively suppressed the proliferation of TNBC cells rather than normal cells in terms of an ATP assay. This preferential antiproliferative function was alleviated by pretreating inhibitors for oxidative stress (N-acetylcysteine (NAC)) and apoptosis (Z-VAD-FMK). Accordingly, the oxidative-stress-related mechanisms were further assessed. StenB caused subG1 and G2/M accumulation but reduced the G1 phase in TNBC cells, while normal cells remained unchanged between the control and StenB treatments. The apoptosis behavior of TNBC cells was suppressed by StenB, whereas that of normal cells was not suppressed according to an annexin V assay. StenB-modulated apoptosis signaling, such as for caspases 3, 8, and 9, was more significantly activated in TNBC than in normal cells. StenB also caused oxidative stress in TNBC cells but not in normal cells according to a flow cytometry assay monitoring reactive oxygen species, mitochondrial superoxide, and their membrane potential. StenB induced greater DNA damage responses (γH2AX and 8-hydroxy-2-deoxyguanosine) in TNBC than in normal cells. All these StenB responses were alleviated by NAC pretreatment. Collectively, StenB modulated oxidative stress responses, leading to the antiproliferation of TNBC cells with little cytotoxicity in normal cells.

Ginger-derived compounds are abundant sources of anticancer natural products. However, the anticancer effects of (E)-3-hydroxy-1-(4′-hydroxy-3′,5′-dimethoxyphenyl)-tetradecan-6-en-5-one (3HDT) have not been examined. This study aims to assess the antiproliferation ability of 3HDT on triple-negative breast cancer (TNBC) cells. 3HDT showed dose-responsive antiproliferation for TNBC cells (HCC1937 and Hs578T). Moreover, 3HDT exerted higher antiproliferation and apoptosis on TNBC cells than on normal cells (H184B5F5/M10). By examining reactive oxygen species, mitochondrial membrane potential, and glutathione, we found that 3HDT provided higher inductions for oxidative stress in TNBC cells compared with normal cells. Antiproliferation, oxidative stress, antioxidant signaling, and apoptosis were recovered by N-acetylcysteine, indicating that 3HDT preferentially induced oxidative-stress-mediated antiproliferation in TNBC cells but not in normal cells. Moreover, by examining γH2A histone family member X (γH2AX) and 8-hydroxy-2-deoxyguanosine, we found that 3HDT provided higher inductions for DNA damage, which was also reverted by N-acetylcysteine. In conclusion, 3HDT is an effective anticancer drug with preferential antiproliferation, oxidative stress, apoptosis, and DNA damage effects on TNBC cells.

The anticancer effects and mechanisms of marine sponge Aaptos suberitoides were rarely assessed, especially for methanol extract of A. suberitoides (MEAS) to breast cancer cells. This study evaluated the differential suppression effects of proliferation by MEAS between breast cancer and normal cells. MEAS demonstrated more antiproliferation impact on breast cancer cells than normal cells, indicating oxidative stress-dependent preferential antiproliferation effects on breast cancer cells but not for normal cells. Several oxidative stress-associated responses were highly induced by MEAS in breast cancer cells but not normal cells, including the generations of cellular and mitochondrial oxidative stress as well as the depletion of mitochondrial membrane potential. MEAS downregulated cellular antioxidants such as glutathione, partly contributing to the upregulation of oxidative stress in breast cancer cells. This preferential oxidative stress generation is accompanied by more DNA damage (γH2AX and 8-hydroxy-2-deoxyguanosine) in breast cancer cells than in normal cells. N-acetylcysteine reverted these MEAS-triggered responses. In conclusion, MEAS is a potential natural product for treating breast cancer cells with the characteristics of preferential antiproliferation function without cytotoxicity to normal cells in vitro.

Physapruin A (PHA), a Physalis peruviana-derived withanolide, exhibits antiproliferation activity against oral and breast cancer cells. However, its potential antitumor effects in combined treatments remain unclear. This investigation focused on evaluating the impact of the combined treatment of ultraviolet-C with PHA (UVC/PHA) on the proliferation of oral cancer cells. The UVC-caused antiproliferation was enhanced by combination with PHA in oral cancer (Ca9-22 and CAL 27) but not normal cells (SG), as evidenced by ATP detection, compared with UVC or PHA alone. UVC/PHA showed a greater extent of subG1 increase, G2/M arrest, annexin-V-assessed apoptosis, caspase 3/7 activation, and reactive oxygen species (ROS) in the UVC or PHA treatment of oral cancer compared to normal cells. Moreover, the mitochondrial functions, such as mitochondrial superoxide bursts and mitochondrial membrane potential destruction, of oral cancer cells were also enhanced by UVC/PHA compared to UVC or PHA alone. These oxidative stresses triggered γH2AX and 8-hydroxyl-2’-deoxyguanosine-assessed DNA damage to a greater extent under UVC/PHA treatment than under UVC or PHA treatment alone. The ROS inhibitor N-acetylcysteine reversed all these UVC/PHA-promoted changes. In conclusion, UVC/PHA is a promising strategy for decreasing the proliferation of oral cancer cells but shows no inhibitory effect on normal cells.

The purpose of this study aimed to assess the antiproliferation effects of methanol extract of T. swinhoei (METS) and explore the detailed responses of oral cancer cells compared to normal cells. METS effectively inhibits the cell proliferation of oral cancer cells but does not affect normal cell viability, exhibiting preferential antiproliferation function. METS exerted more subG1 accumulation, apoptosis induction, cellular and mitochondrial oxidative stress, and DNA damage than normal cells, reverted by oxidative stress inhibitor N-acetylcysteine. This METS-caused oxidative stress was validated to attribute to the downregulation of glutathione. METS activated both extrinsic and intrinsic caspases. DNA double-strand breaks (γH2AX) and oxidative DNA damage (8-hydroxy-2-deoxyguanosine) were stimulated by METS. Therefore, for the first time, this investigation shed light on exploring the functions and responses of preferential antiproliferation of METS in oral cancer cells.

A novel nitrated [6,6,6]tricycles-derived compound containing nitro, methoxy, and ispropyloxy groups, namely SK1, was developed in our previous report. However, the anticancer effects of SK1 were not assessed. Moreover, SK1 contains two nitro groups (NO2) and one nitrogen-oxygen (N-O) bond exhibiting the potential for oxidative stress generation, but this was not examined. The present study aimed to evaluate the antiproliferation effects and oxidative stress and its associated responses between oral cancer and normal cells. Based on the MTS assay, SK1 demonstrated more antiproliferation ability in oral cancer cells than normal cells, reversed by N-acetylcysteine. This suggests that SK1 causes antiproliferation effects preferentially in an oxidative stress-dependent manner. The oxidative stress-associated responses were further validated, showing higher ROS/MitoSOX burst, MMP, and GSH depletion in oral cancer cells than in normal cells. Meanwhile, SK1 caused oxidative stress-causing apoptosis, such as caspases 3/8/9, and DNA damages, such as γH2AX and 8-OHdG, to a greater extent in oral cancer cells than in normal cells. Siilar to cell viability, these oxidative stress responses were partially diminished by NAC, indicating that SK1 promoted oxidative stress-dependent responses. In conclusion, SK1 exerts oxidative stress, apoptosis, and DNA damage to a greater extent to oral cancer cells than in normal cells.

Antiproliferation effects of Clavularia-derived natural products against cancer cells have been reported on, but most studies have focused on identifying bioactive compounds, lacking a detailed investigation of the molecular mechanism. Crude extracts generally exhibit multiple targeting potentials for anticancer effects, but they have rarely been assessed for methanol extracts of Clavularia inflata (MECI). This investigation aims to evaluate the antiproliferation of MECI and to examine several potential mechanisms between oral cancer and normal cells. A 24 h MTS assay demonstrated that MECI decreased cell viability in several oral cancer cell lines more than in normal cells. N-acetylcysteine (NAC), an oxidative stress inhibitor, recovered these antiproliferation effects. Higher oxidative stress was stimulated by MECI in oral cancer cells than in normal cells, as proven by examining reactive oxygen species and mitochondrial superoxide. This preferential induction of oxidative stress was partly explained by downregulating more cellular antioxidants, such as glutathione, in oral cancer cells than in normal cells. Consequently, the MECI-generated high oxidative stress in oral cancer cells was preferred to trigger more subG1 population, apoptosis expression (annexin V and caspase activation), and DNA damage, reverted by NAC. In conclusion, MECI is a potent marine natural product showing preferential antiproliferation against oral cancer cells.

Combined treatment is a promising anticancer strategy for improving antiproliferation compared with a single treatment but is limited by adverse side effects on normal cells. Fucoidan (FN), a brown-algae-derived polysaccharide safe food ingredient, exhibits preferential function for antiproliferation to oral cancer but not normal cells. Utilizing the preferential antiproliferation, the impacts of FN in regulating ultraviolet C (UVC) irradiation were assessed in oral cancer cells. A combined treatment (UVC/FN) reduced cell viability of oral cancer cells (Ca9-22 and CAL 27) more than single treatments (FN or UVC), i.e., 53.7%/54.6% vs. 71.2%/91.6%, and 89.2%/79.4%, respectively, while the cell viability of UVC/FN treating on non-malignant oral (S–G) was higher than oral cancer cells, ranging from 106.0 to 108.5%. Mechanistically, UVC/FN preferentially generated higher subG1 accumulation and apoptosis-related inductions (annexin V, caspases 3, 8, and 9) in oral cancer cells than single treatments. UVC/FN preferentially generated higher oxidative stress than single treatments, as evidenced by flow cytometry-detecting reactive oxygen species, mitochondrial superoxide, and glutathione. Moreover, UVC/FN preferentially caused more DNA damage (γH2AX and 8-hydroxy-2’-deoxyguanosine) in oral cancer cells than in single treatments. N-acetylcysteine pretreatment validated the oxidative stress effects in these UVC/FN-induced changes. Taken together, FN effectively enhances UVC-triggered antiproliferation to oral cancer cells. UVC/FN provides a promising potential for preferential and synergistic antiproliferation in antioral cancer therapy.

Oxidative stress and AKT serine-threonine kinase (AKT) are responsible for regulating several cell functions of cancer cells. Several natural products modulate both oxidative stress and AKT for anticancer effects. However, the impact of natural product-modulating oxidative stress and AKT on cell functions lacks systemic understanding. Notably, the contribution of regulating cell functions by AKT downstream effectors is not yet well integrated. This review explores the role of oxidative stress and AKT pathway (AKT/AKT effectors) on ten cell functions, including apoptosis, autophagy, endoplasmic reticulum stress, mitochondrial morphogenesis, ferroptosis, necroptosis, DNA damage response, senescence, migration, and cell-cycle progression. The impact of oxidative stress and AKT are connected to these cell functions through cell function mediators. Moreover, the AKT effectors related to cell functions are integrated. Based on this rationale, natural products with the modulating abilities for oxidative stress and AKT pathway exhibit the potential to regulate these cell functions, but some were rarely reported, particularly for AKT effectors. This review sheds light on understanding the roles of oxidative stress and AKT pathway in regulating cell functions, providing future directions for natural products in cancer treatment.

The selective antiproliferation to oral cancer cells of Physalis peruviana-derived physapruin A (PHA) is rarely reported. Either drug-induced apoptosis and DNA damage or DNA repair suppression may effectively inhibit cancer cell proliferation. This study examined the selective antiproliferation ability of PHA and explored detailed mechanisms of apoptosis, DNA damage, and repair. During an ATP assay, PHA provided high cytotoxicity to two oral cancer cell lines (CAL 27 and Ca9-22) but no cytotoxicity to two non-malignant oral cells (HGF-1 and SG). This selective antiproliferation of PHA was associated with the selective generation of reactive oxygen species (ROS) in oral cancer cells rather than in non-malignant oral cells, as detected by flow cytometry. Moreover, PHA induced other oxidative stresses in oral cancer cells, such as mitochondrial superoxide generation and mitochondrial membrane potential depletion. PHA also demonstrated selective apoptosis in oral cancer cells rather than non-malignant cells in annexin V/7-aminoactinmycin D and caspase 3/7 activity assays. In flow cytometry and immunofluorescence assays, PHA induced γH2AX expressions and increased the γH2AX foci number of DNA damages in oral cancer cells. In contrast, the mRNA expressions for DNA repair signaling, including homologous recombination (HR) and non-homologous end joining (NHEJ)-associated genes, were inhibited by PHA in oral cancer cells. Moreover, the PHA-induced changes were alleviated by the oxidative stress inhibitor N-acetylcysteine. Therefore, PHA generates selective antiproliferation, oxidative stress, and apoptosis associated with DNA damage induction and DNA repair suppression in oral cancer cells.

Physalis peruviana-derived physapruin A (PHA) is a potent compound that selectively generates reactive oxygen species (ROS) and induces cancer cell death. Autophagy, a cellular self-clearance pathway, can be induced by ROS and plays a dual role in cancer cell death. However, the role of autophagy in PHA-treated cancer cells is not understood. Our study initially showed that autophagy inhibitors such as bafilomycin A1 enhanced the cytotoxic effects of PHA in breast cancer cell lines, including MCF7 and MDA-MB-231. PHA treatment decreased the p62 protein level and increased LC3-II flux. PHA increased the fluorescence intensity of DAPGreen and DALGreen, which are used to reflect the formation of autophagosome/autolysosome and autolysosome, respectively. ROS scavenger N-acetylcysteine (NAC) decreased PHA-elevated autophagy activity, implying that PHA-induced ROS may be required for autophagy induction in breast cancer cells. Moreover, the autophagy inhibitor increased ROS levels and enhanced PHA-elevated ROS levels, while NAC scavenges the produced ROS resulting from PHA and autophagy inhibitor. In addition, the autophagy inhibitor elevated the PHA-induced proportion of annexin V/7-aminoactinmycin D and cleavage of caspase-3/8/9 and poly (ADP-ribose) polymerase. In contrast, NAC and apoptosis inhibitor Z-VAD-FMK blocked the proportion of annexin V/7-aminoactinmycin D and the activation of caspases. Taken together, PHA induced ROS to promote autophagy, which might play an antioxidant and anti-apoptotic role in breast cancer cells.

Excavatolide C (EXCC) is a bioactive compound derived from the gorgonian octocoral Briareum excavatum, and its anticancer effects are rarely addressed, particularly for bladder cancer. This investigation aimed to explore the potential impacts of EXCC on inhibiting the proliferation of three kinds of bladder cancer cells (5637, BFTC905, and T24). EXCC inhibits bladder cancer cell proliferation based on 48 h ATP assay. This antiproliferation function is validated to be oxidative stress dependent. Cellular and mitochondrial oxidative stresses were upregulated by EXCC, accompanied by depleting glutathione and mitochondrial membrane potential. These antiproliferation and oxidative stress events were suppressed by N-acetylcysteine (NAC), indicating that EXCC has an oxidative stress-regulating function for antiproliferation of bladder cancer cells. Oxidative stress-related responses such as apoptosis, caspase activation, and DNA damage were upregulated by EXCC and reverted by NAC. Taken together, the antiproliferation function of EXCC provides a potential treatment against bladder cancer cells via oxidative stress modulation.

Antioral cancer drugs need a greater antiproliferative impact on cancer than on normal cells. Demethoxymurrapanine (DEMU) inhibits proliferation in several cancer cells, but an in‐depth investigation was necessary. This study evaluated the proliferation‐modulating effects of DEMU, focusing on oral cancer and normal cells. DEMU (0, 2, 3, and 4 μg/mL) at 48 h treatments inhibited the proliferation of oral cancer cells (the cell viability (%) for Ca9‐22 cells was 100.0 ± 2.2, 75.4 ± 5.6, 26.0 ± 3.8, and 15.4 ± 1.4, and for CAL 27 cells was 100.0 ± 9.4, 77.2 ± 5.9, 57.4 ± 10.7, and 27.1 ± 1.1) more strongly than that of normal cells (the cell viability (%) for S‐G cells was 100.0 ± 6.6, 91.0 ± 4.6, 95.0 ± 2.6, and 95.8 ± 5.5), although this was blocked by the antioxidant N‐acetylcysteine. The presence of oxidative stress was evidenced by the increase of reactive oxygen species and mitochondrial superoxide and the downregulation of the cellular antioxidant glutathione in oral cancer cells, but these changes were minor in normal cells. DEMU also caused greater induction of the subG1 phase, extrinsic and intrinsic apoptosis (annexin V and caspases 3, 8, and 9), and DNA damage (γH2AX and 8‐hydroxy‐2‐deoxyguanosine) in oral cancer than in normal cells. N‐acetylcysteine attenuated all these DEMU‐induced changes. Together, these data demonstrate the preferential antiproliferative function of DEMU in oral cancer cells, with the preferential induction of oxidative stress, apoptosis, and DNA damage in these cancer cells, and low cytotoxicity toward normal cells. [ABSTRACT FROM AUTHOR]

The anti-oral cancer effects of santamarine (SAMA), a Michelia compressa var. compressa-derived natural product, remain unclear. This study investigates the anticancer effects and acting mechanism of SAMA against oral cancer (OC-2 and HSC-3) in parallel with normal (Smulow–Glickman; S-G) cells. SAMA selectively inhibits oral cancer cell viability more than normal cells, reverted by the oxidative stress remover N-acetylcysteine (NAC). The evidence of oxidative stress generation, such as the induction of reactive oxygen species (ROS) and mitochondrial superoxide and the depletion of mitochondrial membrane potential and glutathione, further supports this ROS-dependent selective antiproliferation. SAMA arrests oral cancer cells at the G2/M phase. SAMA triggers apoptosis (annexin V) in oral cancer cells and activates caspases 3, 8, and 9. SAMA enhances two types of DNA damage in oral cancer cells, such as γH2AX and 8-hydroxy-2-deoxyguanosine. Moreover, all of these anticancer mechanisms of SAMA are more highly expressed in oral cancer cells than in normal cells in concentration and time course experiments. These above changes are attenuated by NAC, suggesting that SAMA exerts mechanisms of selective antiproliferation that depend on oxidative stress while maintaining minimal cytotoxicity to normal cells. [ABSTRACT FROM AUTHOR]

This in vitro study examines the anti-oral cancer effects and mechanisms of a combined X-ray/SK2 treatment, i.e., X-ray and 6-n-butoxy-10-nitro-12,13-dioxa-11-azatricyclo[7.3.1.02,7]trideca-2,4,6,10-tetraene (SK2). ATP cell viability and flow cytometry-based cell cycle, apoptosis, oxidative stress, and DNA damage assessments were conducted. The X-ray/SK2 treatment exhibited lower viability in oral cancer (Ca9-22 and CAL 27) cells than in normal (Smulow–Glickman, S-G) cells, i.e., 32.0%, 46.1% vs. 59.0%, which showed more antiproliferative changes than with X-ray or SK2 treatment. Oral cancer cells under X-ray/SK2 treatment showed slight subG1 and G2/M increments and induced high annexin V-monitored apoptosis compared to X-ray or SK2 treatment. The X-ray/SK2 treatment showed higher caspase 3 and 8 levels for oral cancer cells than other treatments. X-ray/SK2 showed a higher caspase 9 level in CAL 27 cells than other treatments, while Ca9-22 cells showed similar levels under X-ray and/or SK2. The X-ray/SK2 treatment showed higher reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) depletion than other treatments. Meanwhile, the mitochondrial superoxide (MitoSOX) and glutathione levels in X-ray/SK2 treatment did not exhibit the highest rank compared to others. Moreover, oral cancer cells had higher γH2AX and/or 8-hydroxy-2-deoxyguanosine levels from X-ray/SK2 treatment than others. All these measurements for X-ray/SK2 in oral cancer cells were higher than in normal cells and attenuated by N-acetylcysteine. In conclusion, X-ray/SK2 treatment showed ROS-dependent enhanced antiproliferative, apoptotic, and DNA damage effects in oral cancer cells with a lower cytotoxic influence on normal cells. [ABSTRACT FROM AUTHOR]

The critical factors for regulating cancer metabolism are oxidative stress and phosphoinositide-3-kinase/AKT serine-threonine kinase/mechanistic target of the rapamycin kinase (PI3K/AKT/mTOR). However, the metabolic impacts of oxidative stress and PI3K/AKT/mTOR on individual mechanisms such as glycolysis (Warburg effect), pentose phosphate pathway (PPP), fatty acid synthesis, tricarboxylic acid cycle (TCA) cycle, glutaminolysis, and oxidative phosphorylation (OXPHOS) are complicated. Therefore, this review summarizes the individual and interacting functions of oxidative stress and PI3K/AKT/mTOR on metabolism. Moreover, natural products providing oxidative stress and PI3K/AKT/mTOR modulating effects have anticancer potential. Using the example of brown algae-derived fucoidan, the roles of oxidative stress and PI3K/AKT/mTOR were summarized, although their potential functions within diverse metabolisms were rarely investigated. We propose a potential application that fucoidan may regulate oxidative stress and PI3K/AKT/mTOR signaling to modulate their associated metabolic regulations. This review sheds light on understanding the impacts of oxidative stress and PI3K/AKT/mTOR on metabolism and the future direction of metabolism-based cancer therapy of fucoidan.

Fucoidan is a dietary brown algae-derived fucose-rich polysaccharide. However, the anticancer effects of fucoidan for oral cancer treatment remain unclear, particularly in terms of its preferential antiproliferation ability and oxidative-stress-associated responses. This study first evaluated the effects and mechanisms of the preferential antiproliferation of fucoidan between oral cancer and non-malignant oral cells (S–G). In a 48 h MTS assay, fucoidan showed higher antiproliferation in response to five types of oral cancer cells, but not S–G cells, demonstrating preferential antiproliferation of oral cancer cells. Oral cancer cells (Ca9-22 and CAL 27) showing high sensitivity to fucoidan were selected to explore the antiproliferation mechanism compared to S–G cells. Fucoidan showed subG1 accumulation and an annexin V increase in apoptosis, accompanied by caspase 8, 9, and 3 activations in oral cancer cells, but not in S–G cells. Fucoidan increased reactive oxygen species and mitochondrial superoxide levels and decreased cellular glutathione in oral cancer cells compared with S–G cells. These oxidative stress effects were attributed to the downregulation of antioxidant signaling genes (NRF2, TXN, and HMOX1) in oral cancer cells rather than S–G cells. Fucoidan showed DNA damage-inducible effects (γH2AX and 8-hydroxy-2-deoxyguanosine) in oral cancer cells but not in S–G cells. Accordingly, these preferential changes in oral cancer but not in non-malignant cells contribute to the preferential antiproliferation mechanism of fucoidan. Furthermore, these changes were reverted by pretreatment with the antioxidant N-acetylcysteine. Therefore, for the first time, this study provides a detailed understanding of the preferential antiproliferation effects and mechanisms of fucoidan in oral cancer cells.

Ginger is a popular spice and consists of several bioactive antioxidant compounds. Gingerenone A (Gin A), a novel compound isolated from Zingiber officinale, is rarely investigated for its anti-breast-cancer properties. Some ginger extracts have been reported to initiate senescence, an anticancer strategy. However, the anticancer effects of Gin A on breast cancer cells remain unclear. The present study aims to assess the modulating impact of Gin A acting on proliferation and senescence to breast cancer cells. Gin A diminished the cellular ATP content and decreased the cell viability of the MTS assay in several breast cancer cell lines. It also showed a delayed G2/M response to breast cancer cells (MCF7 and MDA-MB-231). N-acetylcysteine (NAC), an oxidative stress inhibitor, can revert these responses of antiproliferation and G2/M delay. The oxidative stress and senescence responses of Gin A were further validated by increasing reactive oxygen species, mitochondrial superoxide, and β-galactosidase activity, which were reverted by NAC. Gin A also upregulated senescence-associated gene expressions. In addition to oxidative stress, Gin A also induced DNA damage responses by increasing γH2AX level and foci and generating 8-hydroxyl-2′-deoxyguanosine in breast cancer cells, which were reverted by NAC. Therefore, Gin A promotes antiproliferation and senescence of breast cancer cells induced by oxidative stress.

Oxidative stress and the AKT serine/threonine kinase (AKT) signaling pathway are essential regulators in cellular migration, metastasis, and angiogenesis. More than 300 withanolides were discovered from the plant family Solanaceae, exhibiting diverse functions. Notably, the relationship between oxidative stress, AKT signaling, and angiogenesis in withanolide treatments lacks comprehensive understanding. Here, we summarize connecting evidence related to oxidative stress, AKT signaling, and angiogenesis in the zebrafish model. A convenient vertebrate model monitored the in vivo effects of developmental and tumor xenograft angiogenesis using zebrafish embryos. The oxidative stress and AKT-signaling-modulating abilities of withanolides were highlighted in cancer treatments, which indicated that further assessments of their angiogenesis-modulating potential are necessary in the future. Moreover, targeting AKT for inhibiting AKT and its AKT signaling shows the potential for anti-migration and anti-angiogenesis purposes for future application to withanolides. This particularly holds for investigating the anti-angiogenetic effects mediated by the oxidative stress and AKT signaling pathways in withanolide-based cancer therapy in the future.

Burmannic acid (BURA) is a new apocarotenoid bioactive compound derived from Indonesian cinnamon; however, its anticancer effect has rarely been investigated in oral cancer cells. In this investigation, the consequences of the antiproliferation of oral cancer cells effected by BURA were evaluated. BURA selectively suppressed cell proliferation of oral cancer cells (Ca9-22 and CAL 27) but showed little cytotoxicity to normal oral cells (HGF-1). In terms of mechanism, BURA perturbed cell cycle distribution, upregulated mitochondrial superoxide, induced mitochondrial depolarization, triggered γH2AX and 8-hydroxy-2-deoxyguanosine DNA damage, and induced apoptosis and caspase 3/8/9 activation in oral cancer cells. Application of N-acetylcysteine confirmed oxidative stress as the critical factor in promoting antiproliferation, apoptosis, and DNA damage in oral cancer cells.

Several kinds of solvents have been applied to Nepenthes extractions exhibiting antioxidant and anticancer effects. However, they were rarely investigated for Nepenthes ethyl acetate extract (EANT), especially leukemia cells. The purpose of the present study was to evaluate the antioxidant properties and explore the antiproliferation impact and mechanism of EANT in leukemia cells. Five standard assays demonstrated that EANT exhibits antioxidant capability. In the cell line model, EANT dose-responsively inhibited cell viabilities of three leukemia cell lines (HL-60, K-562, and MOLT-4) based on 24 h MTS assays, which were reverted by pretreating oxidative stress and apoptosis inhibitors (N-acetylcysteine and Z-VAD-FMK). Due to similar sensitivities among the three cell lines, leukemia HL-60 cells were chosen for exploring antiproliferation mechanisms. EANT caused subG1 and G1 cumulations, triggered annexin V-detected apoptosis, activated apoptotic caspase 3/7 activity, and induced poly ADP-ribose polymerase expression. Moreover, reactive oxygen species, mitochondrial superoxide, and mitochondrial membrane depolarization were generated by EANT, which was reverted by N-acetylcysteine. The antioxidant response to oxidative stress showed that EANT upregulated mRNA expressions for nuclear factor erythroid 2-like 2 (NFE2L2), catalase (CAT), thioredoxin (TXN), heme oxygenase 1 (HMOX1), and NAD(P)H quinone dehydrogenase 1 (NQO1) genes. Moreover, these oxidative stresses led to DNA damage (γH2AX and 8-hydroxy-2-deoxyguanosine) and were alleviated by N-acetylcysteine. Taken together, EANT demonstrated oxidative stress-dependent anti-leukemia ability to HL-60 cells associated with apoptosis and DNA damage.

Ethyl acetate Nepenthes extract (EANT) from Nepenthes thorellii × (ventricosa × maxima) shows antiproliferation and apoptosis but not necrosis in breast cancer cells, but this has not been investigated in oral cancer cells. In the present study, EANT shows no cytotoxicity to normal oral cells but exhibits selective killing to six oral cancer cell lines. They were suppressed by pretreatment of the antioxidant inhibitor N-acetylcysteine (NAC), demonstrating that EANT-induced cell death was mediated by oxidative stress. Concerning high sensitivity to EANT, Ca9-22 and CAL 27 oral cancer cells were chosen for exploring detailed selective killing mechanisms. EANT triggers a mixture of necrosis and apoptosis as determined by annexin V/7-aminoactinmycin D analysis. Still, they show differential switches from necrosis at a low (10 μg/mL) concentration to apoptosis at high (25 μg/mL) concentration of EANT in oral cancer cells. NAC induces necrosis but suppresses annexin V-detected apoptosis in oral cancer cells. Necrostatin 1 (NEC1), a necroptosis inhibitor, moderately suppresses necrosis but induces apoptosis at 10 μg/mL EANT. In contrast, Z-VAD-FMK, a pancaspase inhibitor, slightly causes necrosis but suppresses apoptosis at 10 μg/mL EANT. Furthermore, the flow cytometry-detected pancaspase activity is dose-responsively increased but is suppressed by NAC and ZVAD, although not for NEC1 in oral cancer cells. EANT causes several oxidative stress events such as reactive oxygen species, mitochondrial superoxide, and mitochondrial membrane depolarization. In response to oxidative stresses, the mRNA for antioxidant signaling, such as nuclear factor erythroid 2-like 2 (NFE2L2), catalase (CAT), heme oxygenase 1 (HMOX1), and thioredoxin (TXN), are overexpressed in oral cancer cells. Moreover, EANT also triggers DNA damage, as detected by γH2AX and 8-oxo-2′-deoxyguanosine adducts. The dependence of oxidative stress is validated by the evidence that NAC pretreatment reverts the changes of cellular and mitochondrial stress and DNA damage. Therefore, EANT exhibits antiproliferation involving an oxidative stress-dependent necrosis/apoptosis switch and DNA damage in oral cancer cells.

Withaferin A (WFA), the Indian ginseng bioactive compound, exhibits an antiproliferation effect on several kinds of cancer, but it was rarely reported in bladder cancer cells. This study aims to assess the anticancer effect and mechanism of WFA in bladder cancer cells. WFA shows antiproliferation to bladder cancer J82 cells based on the finding of the MTS assay. WFA disturbs cell cycle progression associated with subG1 accumulation in J82 cells. Furthermore, WFA triggers apoptosis as determined by flow cytometry assays using annexin V/7-aminoactinomycin D and pancaspase detection. Western blotting also supports WFA-induced apoptosis by increasing cleavage of caspases 3, 8, and 9 and poly ADP-ribose polymerase. Mechanistically, WFA triggers oxidative stress-association changes, such as the generation of reactive oxygen species and mitochondrial superoxide and diminishment of the mitochondrial membrane potential, in J82 cells. In response to oxidative stresses, mRNA for antioxidant signaling, such as nuclear factor erythroid 2-like 2 (NFE2L2), catalase (CAT), superoxide dismutase 1 (SOD1), thioredoxin (TXN), glutathione-disulfide reductase (GSR), quinone dehydrogenase 1 (NQO1), and heme oxygenase 1 (HMOX1), are overexpressed in J82 cells. In addition, WFA causes DNA strand breaks and oxidative DNA damages. Moreover, the ROS scavenger N-acetylcysteine reverts all tested WFA-modulating effects. In conclusion, WFA possesses anti-bladder cancer effects by inducing antiproliferation, apoptosis, and DNA damage in an oxidative stress-dependent manner.

Breast cancer expresses clinically heterogeneous characteristics and requires multipurpose drug development for curing the different tumor subtypes. Many withanolides have been isolated from Physalis species showing anticancer effects, but the anticancer function of physapruin A (PHA) has rarely been investigated. In this study, the anticancer properties of PHA in breast cancer cells were examined by concentration and time-course experiments. In terms of cellular ATP content, PHA inhibited the proliferation of three kinds of breast cancer cells: MCF7 (estrogen receptor (ER)+, progesterone receptor (PR)+/−, human epidermal growth factor receptor 2 (HER2)−), SKBR3 (ER−/PR−/HER2+), and MDA-MB-231 (triple-negative). Moreover, PHA induced G2/M arrest in MCF7 and MDA-MB-231 cells. In terms of flow cytometry, PHA induced the generation of reactive oxygen species (ROS), the generation of mitochondrial superoxide, mitochondrial membrane potential depletion, and γH2AX-detected DNA damage in breast cancer MCF7 and MDA-MB-231 cells, which were suppressed by the ROS inhibitor N-acetylcysteine (NAC). In terms of flow cytometry and Western blotting, PHA induced apoptotic expression (annexin V, and intrinsic and extrinsic apoptotic signaling), which was suppressed by NAC and an apoptosis inhibitor (Z-VAD-FMK), in breast cancer cells. Therefore, PHA is a potential anti-breast-cancer natural product that modulates the oxidative-stress response, cell-cycle disturbance, apoptosis, and γH2AX-detected DNA damage.

Some withanolides, particularly the family of steroidal lactones, show anticancer effects, but this is rarely reported for withanolide C (WHC)—especially anti-breast cancer effects. The subject of this study is to evaluate the ability of WHC to regulate the proliferation of breast cancer cells, using both time and concentration in treatment with WHC. In terms of ATP depletion, WHC induced more antiproliferation to three breast cancer cell lines, SKBR3, MCF7, and MDA-MB-231, than to normal breast M10 cell lines. SKBR3 and MCF7 cells showing higher sensitivity to WHC were used to explore the antiproliferation mechanism. Flow cytometric apoptosis analyses showed that subG1 phase and annexin V population were increased in breast cancer cells after WHC treatment. Western blotting showed that cleaved forms of the apoptotic proteins poly (ADP-ribose) polymerase (c-PARP) and cleaved caspase 3 (c-Cas 3) were increased in breast cancer cells. Flow cytometric oxidative stress analyses showed that WHC triggered reactive oxygen species (ROS) and mitochondrial superoxide (MitoSOX) production as well as glutathione depletion. In contrast, normal breast M10 cells showed lower levels of ROS and annexin V expression than breast cancer cells. Flow cytometric DNA damage analyses showed that WHC triggered γH2AX and 8-oxo-2′-deoxyguanosine (8-oxodG) expression in breast cancer cells. Moreover, N-acetylcysteine (NAC) pretreatment reverted oxidative stress-mediated ATP depletion, apoptosis, and DNA damage. Therefore, WHC kills breast cancer cells depending on oxidative stress-associated mechanisms.

Some lichens provide the resources of common traditional medicines and show anticancer effects. However, the anticancer effect of Usnproliea barbata (U. barbata) is rarely investigated, especially for oral cancer cells. The aim of this study was to investigate the cell killing function of methanol extracts of U. barbata (MEUB) against oral cancer cells. MEUB shows preferential killing against a number of oral cancer cell lines (Ca9-22, OECM-1, CAL 27, HSC3, and SCC9) but rarely affects normal oral cell lines (HGF-1). Ca9-22 and OECM-1 cells display the highest sensitivity to MEUB and were chosen for concentration effect and time course experiments to address its cytotoxic mechanisms. MEUB induces apoptosis of oral cancer cells in terms of the findings from flow cytometric assays and Western blotting, such as subG1 accumulation, annexin V detection, and pancaspase activation as well as poly (ADP-ribose) polymerase (PARP) cleavage. MEUB induces oxidative stress and DNA damage of oral cancer cells following flow cytometric assays, such as reactive oxygen species (ROS)/mitochondrial superoxide (MitoSOX) production, mitochondrial membrane potential (MMP) depletion as well as overexpression of γH2AX and 8-oxo-2′deoxyguanosine (8-oxodG). All MEUB-induced changes in oral cancer cells were triggered by oxidative stress which was validated by pretreatment with antioxidant N-acetylcysteine (NAC). In conclusion, MEUB causes preferential killing of oral cancer cells and is associated with oxidative stress, apoptosis, and DNA damage.

Withaferin A (WFA) has been reported to inhibit cancer cell proliferation based on high cytotoxic concentrations. However, the low cytotoxic effect of WFA in regulating cancer cell migration is rarely investigated. The purpose of this study is to investigate the changes in migration and mechanisms of oral cancer Ca9-22 cells after low concentrations of WFA treatment. WFA under 0.5 μM at 24 h treatment shows no cytotoxicity to oral cancer Ca9-22 cells (~95% viability). Under this condition, WFA triggers reactive oxygen species (ROS) production and inhibits 2D (wound healing) and 3D cell migration (transwell) and Matrigel invasion. Mechanically, WFA inhibits matrix metalloproteinase (MMP)-2 and MMP-9 activities but induces mRNA expression for a group of antioxidant genes, such as nuclear factor, erythroid 2-like 2 (NFE2L2), heme oxygenase 1 (HMOX1), glutathione-disulfide reductase (GSR), and NAD(P)H quinone dehydrogenase 1 (NQO1)) in Ca9-22 cells. Moreover, WFA induces mild phosphorylation of the mitogen-activated protein kinase (MAPK) family, including extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 expression. All WFA-induced changes were suppressed by the presence of ROS scavenger N-acetylcysteine (NAC). Therefore, these results suggest that low concentration of WFA retains potent ROS-mediated anti-migration and -invasion abilities for oral cancer cells.

Physalis plants are commonly used traditional medicinal herbs, and most of their extracts containing withanolides show anticancer effects. Physapruin A (PHA), a withanolide isolated from P. peruviana, shows antiproliferative effects on breast cancer cells involving oxidative stress, apoptosis, and autophagy. However, the other oxidative stress-associated response, such as endoplasmic reticulum (ER) stress, and its participation in regulating apoptosis in PHA-treated breast cancer cells remain unclear. This study aims to explore the function of oxidative stress and ER stress in modulating the proliferation and apoptosis of breast cancer cells treated with PHA. PHA induced a more significant ER expansion and aggresome formation of breast cancer cells (MCF7 and MDA-MB-231). The mRNA and protein levels of ER stress-responsive genes (IRE1α and BIP) were upregulated by PHA in breast cancer cells. The co-treatment of PHA with the ER stress-inducer (thapsigargin, TG), i.e., TG/PHA, demonstrated synergistic antiproliferation, reactive oxygen species generation, subG1 accumulation, and apoptosis (annexin V and caspases 3/8 activation) as examined by ATP assay, flow cytometry, and western blotting. These ER stress responses, their associated antiproliferation, and apoptosis changes were partly alleviated by the N-acetylcysteine, an oxidative stress inhibitor. Taken together, PHA exhibits ER stress-inducing function to promote antiproliferation and apoptosis of breast cancer cells involving oxidative stress. [ABSTRACT FROM AUTHOR]

Triple-negative breast cancer (TNBC) is insensitive to target therapy for non-TNBC and needs novel drug discovery. Extracts of the traditional herb Boesenbergia plant in Southern Asia exhibit anticancer effects and contain novel bioactive compounds but merely show cytotoxicity. We recently isolated a new compound from B. stenophylla, stenophyllol B (StenB), but the impact and mechanism of its proliferation-modulating function on TNBC cells remain uninvestigated. This study aimed to assess the antiproliferative responses of StenB in TNBC cells and examine the drug safety in normal cells. StenB effectively suppressed the proliferation of TNBC cells rather than normal cells in terms of an ATP assay. This preferential antiproliferative function was alleviated by pretreating inhibitors for oxidative stress (N-acetylcysteine (NAC)) and apoptosis (Z-VAD-FMK). Accordingly, the oxidative-stress-related mechanisms were further assessed. StenB caused subG1 and G2/M accumulation but reduced the G1 phase in TNBC cells, while normal cells remained unchanged between the control and StenB treatments. The apoptosis behavior of TNBC cells was suppressed by StenB, whereas that of normal cells was not suppressed according to an annexin V assay. StenB-modulated apoptosis signaling, such as for caspases 3, 8, and 9, was more significantly activated in TNBC than in normal cells. StenB also caused oxidative stress in TNBC cells but not in normal cells according to a flow cytometry assay monitoring reactive oxygen species, mitochondrial superoxide, and their membrane potential. StenB induced greater DNA damage responses (γH2AX and 8-hydroxy-2-deoxyguanosine) in TNBC than in normal cells. All these StenB responses were alleviated by NAC pretreatment. Collectively, StenB modulated oxidative stress responses, leading to the antiproliferation of TNBC cells with little cytotoxicity in normal cells. [ABSTRACT FROM AUTHOR]

Withaferin A (WFA) is one of the most active steroidal lactones with reactive oxygen species (ROS) modulating effects against several types of cancer. ROS regulation involves selective killing. However, the anticancer and selective killing effects of WFA against oral cancer cells remain unclear. We evaluated whether the killing ability of WFA is selective, and we explored its mechanism against oral cancer cells. An MTS tetrazolium cell proliferation assay confirmed that WFA selectively killed two oral cancer cells (Ca9-22 and CAL 27) rather than normal oral cells (HGF-1). WFA also induced apoptosis of Ca9-22 cells, which was measured by flow cytometry for subG1 percentage, annexin V expression, and pan-caspase activity, as well as western blotting for caspases 1, 8, and 9 activations. Flow cytometry analysis shows that WFA-treated Ca9-22 oral cancer cells induced G2/M cell cycle arrest, ROS production, mitochondrial membrane depolarization, and phosphorylated histone H2A.X (γH2AX)-based DNA damage. Moreover, pretreating Ca9-22 cells with N-acetylcysteine (NAC) rescued WFA-induced selective killing, apoptosis, G2/M arrest, oxidative stress, and DNA damage. We conclude that WFA induced oxidative stress-mediated selective killing of oral cancer cells.