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Bisphenol A (BPA) is a ubiquitous environmental pollutant with endocrine-disrupting functions. Identifying protective drugs and exploring the mechanisms against BPA are crucial in healthcare. Natural products exhibiting antioxidant properties are considered to be able to protect against BPA toxicity. Although BPA-modulated targets and miRNAs have been individually reported, their connections to natural products were rarely organized. With the help of a protein–protein interaction database (STRING), the relationship between individual BPA-modulated targets was interconnected to provide a systemic view. In this review, BPA-downregulated and -upregulated targets are classified, and their interactive network was innovatively analyzed using the bioinformatic database (STRING). BPA-modulated miRNAs were also retrieved and ingeniously connected to BPA-modulated targets. Moreover, a novel connection between BPA-countering natural products was integrated into BPA-modulated miRNAs and targets. All these targets-associated natural products and/or miRNAs were incorporated into the STRING network, providing systemic relationships. Overall, the BPA-modulated target–miRNA-protecting natural product axis was innovatively constructed, providing a straightforward direction for exploring the integrated BPA-countering effects and mechanisms of natural products.

Immunogenic cell death (ICD) spatiotemporally regulates damage-associated molecular patterns (DAMPs) derived from dying cancer cells to signal the immune response. Intriguingly, these DAMPs and cytokines also induce cellular responses in non-immune cells, particularly cancer cells. Several ICD-modulating natural products and miRNAs have been reported to regulate the DAMP, cytokine, and cell death responses, but they lack systemic organization and connection. This review summarizes the impacts of natural products and miRNAs on the DAMP and cytokine responses and cancer cell death responses (apoptosis, autophagy, ferroptosis, necroptosis, and pyroptosis). We establish the rationale that ICD inducers of natural products have modulating effects on miRNAs, targeting DAMPs and cytokines for immune and cancer cell death responses. In conclusion, DAMP, cytokine, and cell death responses are intricately linked in cancer cells, and they are influenced by ICD-modulating natural products and miRNAs.

Background/Purpose: High-dose-rate (HDR) electronic brachytherapy (EBT) has been shown to be effective for non-melanoma skin cancer (NMSC) in Caucasian patients. However, its efficacy remains unknown in Asian patients. To analyze the clinical outcome of HDR EBT for NMSC in a Taiwanese medical center. Methods: Medical records over a 5-year period between January 2015 to December 2019 were retrospectively analyzed. Results: Forty-seven patients with 54 NMSC including 42 basal cell carcinomas (BCCs) and 12 squamous cell carcinomas (SCCs) were treated with HDR EBT. The average age was 73.8 years. The mean radiation dose was 45.3 Gy (40–80 Gy). Mean follow-up duration was 33.1 months. Adequate local control was achieved in 50 lesions (92.6%). Grade 1 acute skin toxicity was noted in 63.0% of lesions, while no tumors had Grade 4 acute toxicity. No ulceration was observed six months after completion of treatment. At the last follow-up visit, all lesions were rated to have “fair” to “excellent” cosmetic outcomes. Conclusion: HDR EBT provides adequate clinical outcomes and cosmetic results for NMSC in Asian patients. Further investigation of the dosage guidelines is needed for Asian patients with NMSC.

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.

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.

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.

Data regarding the effects of crude extract of Commelina plants in oral cancer treatment are scarce. This present study aimed to assess the proliferation-modulating effects of the Commelina sp. (MECO) methanol extract on oral cancer cells in culture, Ca9-22, and CAL 27. MECO suppressed viability to a greater extent in oral cancer cells than in normal cells. MECO also induced more annexin V, apoptosis, and caspase signaling for caspases 3/8/9 in oral cancer cells. The preferential antiproliferation and apoptosis were associated with cellular and mitochondrial oxidative stress in oral cancer cells. Moreover, MECO also preferentially induced DNA damage in oral cancer cells by elevating γH2AX and 8-hydroxyl-2′-deoxyguanosine. The oxidative stress scavengers N-acetylcysteine or MitoTEMPO reverted these preferential antiproliferation mechanisms. It can be concluded that MECO is a natural product with preferential antiproliferation effects and exhibits an oxidative stress-associated mechanism in oral cancer cells.

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.

AKT serine-threonine kinase (AKT) and its effectors are essential for maintaining cell proliferation, apoptosis, autophagy, endoplasmic reticulum (ER) stress, mitochondrial morphogenesis (fission/fusion), ferroptosis, necroptosis, DNA damage response (damage and repair), senescence, and migration of cancer cells. Several lncRNAs and circRNAs also regulate the expression of these functions by numerous pathways. However, the impact on cell functions by lncRNAs and circRNAs regulating AKT and its effectors is poorly understood. This review provides comprehensive information about the relationship of lncRNAs and circRNAs with AKT on the cell functions of cancer cells. the roles of several lncRNAs and circRNAs acting on AKT effectors, such as FOXO, mTORC1/2, S6K1/2, 4EBP1, SREBP, and HIF are explored. To further validate the relationship between AKT, AKT effectors, lncRNAs, and circRNAs, more predicted AKT- and AKT effector-targeting lncRNAs and circRNAs were retrieved from the LncTarD and circBase databases. Consistently, using an in-depth literature survey, these AKT- and AKT effector-targeting database lncRNAs and circRNAs were related to cell functions. Therefore, some lncRNAs and circRNAs can regulate several cell functions through modulating AKT and AKT effectors. This review provides insights into a comprehensive network of AKT and AKT effectors connecting to lncRNAs and circRNAs in the regulation of cancer cell functions.

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.

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.

SK2, a nitrated [6,6,6]tricycle derivative with an n-butyloxy group, showed selective antiproliferation effects on oral cancer but not on normal oral cells. This investigation assessed for the first time the synergistic antiproliferation potential of cisplatin/SK2 in oral cancer cells. Cell viability assay at 24 h showed that a low dose of combined cisplatin/SK2 (10 μM/10 μg/mL) provided more antiproliferation than cisplatin or SK2 alone. Cisplatin/SK2 triggered also more apoptosis inductions in terms of subG1 accumulation, annexin V, pancaspase, and caspase 3/8/9 measurements. Moreover, cisplatin/SK2 provided more oxidative stress and DNA damage in oral cancer cells than independent treatments. Oxidative stress inhibitors rescued the cisplatin/SK2-induced antiproliferation and oxidative stress generation. Moreover, cisplatin/SK2 induced more antiproliferation, apoptosis, oxidative stress, and DNA damage in oral cancer cells than in normal oral cells (S-G). In conclusion, low-dose cisplatin/SK2 combined treatment promoted selective and synergistic antiproliferation in oral cancer cells depending on oxidative-stress-associated responses.

Combined treatment is an effective strategy to improve anticancer therapy, but severe side effects frequently limit this application. Drugs inhibiting the proliferation of cancer cells, but not normal cells, display preferential antiproliferation to cancer cells. It shows the benefits of avoiding side effects and enhancing antiproliferation for combined treatment. Nitrated [6,6,6]tricycles derivative (SK2), a novel chemical exhibiting benzo-fused dioxabicyclo[3.3.1]nonane core with an n-butyloxy substituent, exhibiting preferential antiproliferation, was chosen to evaluate its potential antioral cancer effect in vitro by combining it with ultraviolet C (UVC) irradiation. Combination treatment (UVC/SK2) caused lower viability in oral cancer cells (Ca9-22 and OC-2) than single treatment (20 J/m2 UVC or 10 μg/mL SK2), i.e., 42.3%/41.1% vs. 81.6%/69.2%, and 89.5%/79.6%, respectively. In contrast, it showed a minor effect on cell viability of normal oral cells (HGF-1), ranging from 82.2 to 90.6%. Moreover, UVC/SK2 caused higher oxidative stress in oral cancer cells than normal cells through the examination of reactive oxygen species, mitochondrial superoxide, and mitochondrial membrane potential. UVC/SK2 also caused subG1 increment associated with apoptosis detections by assessing annexin V; panaspase; and caspases 3, 8, and 9. The antiproliferation and oxidative stress were reverted by N-acetylcysteine, validating the involvement of oxidative stress in antioral cancer cells. UVC/SK2 also caused DNA damage by detecting γH2AX and 8-hydroxy-2′-deoxyguanosine in oral cancer cells. In conclusion, SK2 is an effective enhancer for improving the UVC-caused antiproliferation against oral cancer cells in vitro. UVC/SK2 demonstrated a preferential and synergistic antiproliferation ability towards oral cancer cells with little adverse effects on normal cells.

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.

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.

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.

The benzo-fused dioxabicyclo[3.3.1]nonane core is the central framework in several natural products. Using this core, we had developed a novel nitrated [6,6,6]tricycle-derived compound containing an n-butyloxy group, namely, SK2. The anticancer potential of SK2 was not assessed. This study aimed to determine the antiproliferative function and investigated possible mechanisms of SK2 acting on oral cancer cells. SK2 preferentially killed oral cancer cells but caused no harmful effect on non-malignant oral cells. After the SK2 exposure of oral cancer cells, cells in the sub-G1 phase accumulated. This apoptosis-like outcome of SK2 treatment was validated to be apoptosis via observing an increasing annexin V population. Mechanistically, apoptosis signalers such as pancaspase, caspases 8, caspase 9, and caspase 3 were activated by SK2 in oral cancer cells. SK2 induced oxidative-stress-associated changes. Furthermore, SK2 caused DNA damage (γH2AX and 8-hydroxy-2′-deoxyguanosine). In conclusion, a novel nitrated [6,6,6]tricycle-derived compound, SK2, exhibits a preferential antiproliferative effect on oral cancer cells, accompanied by apoptosis, oxidative stress, and DNA damage.

Dihydrosinularin (DHS) is an analog of soft coral-derived sinularin; however, the anticancer effects and mechanisms of DHS have seldom been reported. This investigation examined the antiproliferation ability and mechanisms of DHS on oral cancer cells. In a cell viability assay, DHS showed growth inhibition against several types of oral cancer cell lines (Ca9-22, SCC-9, OECM-1, CAL 27, OC-2, and HSC-3) with no cytotoxic side effects on non-malignant oral cells (HGF-1). Ca9-22 and SCC-9 cell lines showing high susceptibility to DHS were selected to explore the antiproliferation mechanisms of DHS. DHS also causes apoptosis as detected by annexin V, pancaspase, and caspase 3 activation. DHS induces oxidative stress, leading to the generation of reactive oxygen species (ROS)/mitochondrial superoxide (MitoSOX) and mitochondrial membrane potential (MitoMP) depletion. DHS also induced DNA damage by probing γH2AX phosphorylation. Pretreatment with the ROS scavenger N-acetylcysteine (NAC) can partly counter these DHS-induced changes. We report that the marine natural product DHS can inhibit the cell growth of oral cancer cells. Exploring the mechanisms of this cancer cell growth inhibition, we demonstrate the prominent role DHS plays in oxidative stress.

The anticancer effect of pomegranate polyphenolic extract POMx in oral cancer cells has rarely been explored, especially where its impact on mitochondrial functioning is concerned. Here, we attempt to evaluate the proliferation modulating function and mechanism of POMx against human oral cancer (Ca9-22, HSC-3, and OC-2) cells. POMx induced ATP depletion, subG1 accumulation, and annexin V/Western blotting-detected apoptosis in these three oral cancer cell lines but showed no toxicity to normal oral cell lines (HGF-1). POMx triggered mitochondrial membrane potential (MitoMP) disruption and mitochondrial superoxide (MitoSOX) generation associated with the differential downregulation of several antioxidant gene mRNA/protein expressions in oral cancer cells. POMx downregulated mitochondrial mass, mitochondrial DNA copy number, and mitochondrial biogenesis gene mRNA/protein expression in oral cancer cells. Moreover, POMx induced both PCR-based mitochondrial DNA damage and γH2AX-detected nuclear DNA damage in oral cancer cells. In conclusion, POMx provides antiproliferation and apoptosis of oral cancer cells through mechanisms of mitochondrial impairment.

Marine natural products are abundant resources for antioxidants, but the antioxidant property of the soft corals-derived sinularin and dihydrosinularin were unknown. This study aimed to assess antioxidant potential and antiproliferation effects of above compounds on cancer cells, and to investigate the possible relationships between them. Results show that sinularin and dihydrosinularin promptly reacted with 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS), and hydroxyl (•OH), demonstrating a general radical scavenger activity. Sinularin and dihydrosinularin also show an induction for Fe+3-reduction and Fe+2-chelating capacity which both strengthen their antioxidant activities. Importantly, sinularin shows higher antioxidant properties than dihydrosinularin. Moreover, 24 h ATP assays show that sinularin leads to higher antiproliferation of breast, lung, and liver cancer cells than dihydrosinularin. Therefore, the differential antioxidant properties of sinularin and dihydrosinularin may contribute to their differential anti-proliferation of different cancer cells.

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.

Withaferin A (WFA), a Withania somnifera-derived triterpenoid, is an anticancer natural product. The anticancer effect of nonionizing radiation such as ultraviolet-C (UVC) as well as the combined treatment of UVC and WFA is rarely investigated. Low dose UVC and/or WFA treatments (12 J/m2 and/or 1 μM) were chosen to evaluate antioral cancer cell line effects by examining cytotoxicity, cell cycle disruption, apoptosis induction, and DNA damage. For two cancer cell lines (Ca9-22 and HSC-3), single treatment (UVC or WFA) showed about 80% viability, while a combined treatment of UVC/WFA showed about 40% viability. In contrast, there was noncytotoxicity to normal oral cell lines (HGF-1). Compared to single treatment and control, low dose UVC/WFA shows high inductions of apoptosis in terms of flow cytometric detections for subG1, annexin V, pancaspase changes as well as Western blotting for detecting cleaved poly (ADP-ribose) polymerase (c-PARP) and caspase 3 (c-Cas 3) and luciferase assay for detecting Cas 3/7 activity. Low dose UVC/WFA also showed high inductions of oxidative stress and DNA damage in terms of flow cytometric detections of reactive oxygen species (ROS), mitochondrial superoxide (MitoSOX) generation, and membrane potential (MitoMP) destruction, γH2AX and 8-oxo-2’deoxyguanosine (8-oxodG) types of DNA damages. For comparison, low dose UVC/WFA show rare inductions of annexin V, Cas 3/7 activity, ROS, MitoSOX, and MitoMP changes to normal oral HGF-1 cells. Therefore, low dose UVC/WFA provides a novel selectively killing mechanism to oral cancer cells, suggesting that WFA is a UVC sensitizer to inhibit the proliferation of oral cancer cells.

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.

Ultraviolet-C (UVC) irradiation provides an alternative radiotherapy to X-ray. UVC sensitizer from natural products may improve radiotherapy at low cytotoxic side effects. The aim of this study is to assess the regulation for oral cancer cell proliferation by a combined treatment of UVC and our previously reported anti-oral cancer natural product (ethyl acetate extract of Nepenthes adrianii × clipeata; EANA). The detailed possible UVC sensitizing mechanisms of EANA such as effects on cell proliferation, cell cycle, apoptosis, and DNA damage are investigated individually and in combination using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTS) assay, flow cytometry, and western blotting at low dose conditions. In a 24 h MTS assay, the low dose EANA (5 μg/mL) and low dose UVC (12 J/m2) individually show 80% and combinedly 57% cell proliferation in oral cancer Ca9-22 cells; but no cytotoxicity to normal oral HGF-1 cells. Mechanistically, low dose EANA and low dose UVC individually induce apoptosis (subG1 accumulation, pancaspase activation, and caspases 3, 8, 9), oxidative stress (reactive oxygen species, mitochondrial superoxide, and mitochondrial membrane potential depletion), and DNA damage (γH2AX and 8-hydroxy-2′-deoxyguanosine). Moreover, the combined treatment (UVC/EANA) synergistically induces these changes. Combined low dose treatment-induced antiproliferation, apoptosis, oxidative stress, and DNA damage were suppressed by the ROS scavenger N-acetylcysteine. In conclusion, UVC/EANA shows synergistic antiproliferation, oxidative stress, apoptosis, and DNA damage to oral cancer cells in an oxidative stress-dependent manner. With the selective killing properties of low dose EANA and low dose UVC, EANA provides a novel UVC sensitizing agent to improve the anti-oral cancer therapy.

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.

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.

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.

It is becoming more understandable that an existing challenge for translational research is the development of pharmaceuticals that appropriately target reactive oxygen species (ROS)-mediated molecular networks in cancer cells. In line with this approach, there is an overwhelmingly increasing list of many non-marine drugs and marine drugs reported to be involved in inhibiting and suppressing cancer progression through ROS-mediated cell death. In this review, we describe the strategy of oxidative stress-based therapy and connect the ROS modulating effect to the regulation of apoptosis and autophagy. Finally, we focus on exploring the function and mechanism of cancer therapy by the autophagy modulators including inhibitors and inducers from non-marine drugs and marine drugs.

The water extract of Gracilaria tenuistipitata have been found to be protective against oxidative stress-induced cellular DNA damage, but the biological function of the ethanolic extracts of G. tenuistipitata (EEGT) is still unknown. In this study, the effect of EEGT on oral squamous cell cancer (OSCC) Ca9-22 cell line was examined in terms of the cell proliferation and oxidative stress responses. The cell viability of EEGT-treated OSCC cells was significantly reduced in a dose-response manner (p < 0.0001). The annexin V intensity and pan-caspase activity of EEGT-treated OSCC cells were significantly increased in a dose-response manner (p < 0.05 to 0.0001). EEGT significantly increased the reactive oxygen species (ROS) level (p < 0.0001) and decreased the glutathione (GSH) level (p < 0.01) in a dose-response manner. The mitochondrial membrane potential (MMP) of EEGT-treated OSCC cells was significantly decreased in a dose-response manner (p < 0.005). In conclusion, we have demonstrated that EEGT induced the growth inhibition and apoptosis of OSCC cells, which was accompanied by ROS increase, GSH depletion, caspase activation, and mitochondrial depolarization. Therefore, EEGT may have potent antitumor effect against oral cancer cells.

Isocitrate dehydrogenase (IDH) encodes a nicotinamide adenine dinucleotide phosphate+-dependent enzyme for oxidative decarboxylation of isocitrate and has an essential role in the tricarboxylic acid cycle. Mutations of IDH1 and IDH2 have been identified in patients with glioma, leukemia, and other cancers. However, the incidence of IDH mutations in acute myeloid leukemia in Taiwan is much lower than that reported in Western countries. The reason for the difference is unknown and its clinical implications remain unclear. Acute lymphoblastic leukemia (ALL) is a heterogenous hematopoietic malignancy. Oral squamous cell carcinoma (OSCC) results from chronic carcinogen exposures and is highly prevalent in trucking workers, especially in southern Taiwan. Subtypes of both diseases require specific treatments, and molecular markers for developing tailored treatments are limited. High-resolution melting (HRM) analysis is now a widely used methodology for rapid, accurate, and low-cost mutation scanning. In this study, 90 adults with OSC and 31 children with ALL were scanned by HRM analysis for IDH1 and IDH2 mutation hot spots. In ALL, the allele frequency was 3.23% in both IDH1 and IDH2. In OSCC, the allele frequency was 2.22% in IDH2. A synonymous mutation over pG313 (c.939A > G) of IDH2 was found in both pediatric ALL and adult OSCC. Therefore, we concluded that mutations of IDH are uncommon in ALL and OSCC and are apparently not a major consideration when selecting treatment modalities.

The authors wish to make the following correction to their paper [1].[...]

The natural compound sinularin, isolated from marine soft corals, is antiproliferative against several cancers, but its possible selective killing effect has rarely been investigated. This study investigates the selective killing potential and mechanisms of sinularin-treated breast cancer cells. In 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium, inner salt (MTS) assay, sinularin dose-responsively decreased the cell viability of two breast cancer (SKBR3 and MDA-MB-231) cells, but showed less effect on breast normal (M10) cells after a 24 h treatment. According to 7-aminoactinomycin D (7AAD) flow cytometry, sinularin dose-responsively induced the G2/M cycle arrest of SKBR3 cells. Sinularin dose-responsively induced apoptosis on SKBR3 cells in terms of a flow cytometry-based annexin V/7AAD assay and pancaspase activity, as well as Western blotting for cleaved forms of poly(ADP-ribose) polymerase (PARP), caspases 3, 8, and 9. These caspases and PARP activations were suppressed by N-acetylcysteine (NAC) pretreatment. Moreover, sinularin dose-responsively induced oxidative stress and DNA damage according to flow cytometry analyses of reactive oxygen species (ROS), mitochondrial membrane potential (MitoMP), mitochondrial superoxide, and 8-oxo-2′-deoxyguanosine (8-oxodG)). In conclusion, sinularin induces selective killing, G2/M arrest, apoptosis, and oxidative DNA damage of breast cancer cells.

This study aimed to investigate if zinc compound would have effects on body weight loss and bone marrow suppression induced by total body irradiation (TBI). ICR mice were divided randomly into two groups and treated with test or control compounds. The test compound contained zinc (amino acid chelated with bovine prostate extract), and the control was reverse osmosis pure water (RO water). One week after receiving the treatment, mice were unirradiated, or irradiated with 6 or 3 Gy by 6MV photon beams to the total body. Body weight changes were examined at regular intervals. Three and 5 weeks after the radiation, animals were sacrificed to examine the histologic changes in the bone marrow. Lower body weight in the period of 1-5 weeks after radiation and poor survival rate were found after the 6 Gy TBI, as compared with the 3 Gy groups. The median survival time after 6 Gy and 3 Gy TBI for mice given the test compound were 26 and 76 days, respectively, and the corresponding figures were 14 and 70 days, respectively, for mice given the control compound (p < 0.00001). With zinc supplement, the mean body weight in mice which received the same dose of radiation was 7-8 g heavier than in the water-supplement groups during the second and third weeks (p < 0.05). Hence, there was no statistically significant difference in survival rate between zinc and water supplement in mice given the same dose of irradiation. Histopathologically there was less recovery of bone marrow cells in the 6Gy groups compared with the 3Gy groups. In the 3 Gy water-supplement group, the nucleated cells and megakaryocytes were recovered in the fifth week when recovery was still not seen in the 6Gy group. With zinc supplement, these cells were recovered in the third week. In this study, we found that zinc is beneficial to body weight in mice treated with TBI. Histologic examination of bone marrow showed better recovery of bone marrow cells in groups of mice fed with zinc. This study suggests that zinc can be used as supplements in cancer patients receiving radiotherapy to reduce radiation-induced complications.