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Background: Lactucin, a naturally occurring active sesquiterpene lactone, is abundantly found in chicory and romaine lettuce. A recent study reported that lactucin could induce apoptosis in leukemia cells. However, its cytotoxicity and potential molecular mechanisms underlying cancer cell death remain unclear., Objective: Therefore, in this study, we aimed to investigate the direct effect and underlying mechanism of action of lactucin on renal cancer cells., Methods: MTT assay and flow cytometry were performed to evaluate the rate of cell proliferation and apoptosis, respectively. Western blotting, reverse transcription polymerase chain reaction, and protein stability analyses were performed to analyze the effect of lactucin on the expression of apoptosis-related proteins such as B-cell lymphoma 2 (BCL-2) and CFLAR (CASP8 and FADD like apoptosis regulator) long isoform (CFLAR L ) in Caki-1 human renal cancer cells. In addition, reactive oxygen species (ROS) generation was evaluated using flow cytometry., Results: Lactucin treatment induced apoptosis in Caki-1 cells in a dose-dependent manner via activation of the caspase pathway. It downregulated BCL-2 and CFLAR L expression levels by suppressing BCL-2 transcription and CFLAR L protein stability, respectively. Pretreatment with N-acetyl-1-cysteine, a ROS scavenger, attenuated the lactucin-induced apoptosis and restored the BCL-2 and CFLAR L expression to basal levels. Lactucin-facilitated BCL-2 downregulation was regulated at the transcriptional level through the inactivation of the NF-κB pathway., Conclusions: Our study is the first to demonstrate that lactucin-induced apoptosis is mediated by ROS production, which in turn activates the caspase-dependent apoptotic pathway by inhibiting BCL-2 and CFLAR L expression in Caki-1 cells., (© 2021. The Genetics Society of Korea.)

Neferine is an alkaloid extracted from a seed embryo of Nelumbo nucifera and has recently been shown to have anticancer effects in various human cancer cell lines. However, the detailed molecular mechanism of neferine-induced apoptosis has not been elucidated in renal cancer cells. In the present study, we observed that neferine induced inhibition of cell proliferation and apoptosis in Caki-1 cells in a dose-dependent manner by using MT assay and flow cytometry and that neferine-mediated apoptosis was attenuated by pretreatment with N-benzyloxycarbony-Val-Ala-Asp (O-methyl)-fluoromethyketone, a pan-caspase inhibitor. Treatments with neferine dose-dependently downregulated B cell lymphoma-2 (Bcl-2) expression at the transcriptional level determined by reverse transcriptase-polymerase chain reaction. The forced expression of Bcl-2 and p65 attenuated the neferine-mediated apoptosis in Caki-1 cells. In addition, neferine induced apoptosis by downregulating Bcl-2 and p65 expression in the other two kidney cancer cell lines determined by flow cytometry and western blot analysis. Finally, we observed that treatment with neferine induced apoptosis by inhibiting the NF-κB pathway through caspase-mediated cleavage of the p65 protein by western blot analysis. Collectively, this study demonstrated that neferine-induced apoptosis is mediated by the downregulation of Bcl-2 expression via repression of the NF-κB pathway in renal cancer cells., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Host factors such as nutritional status and immune cell state are important for vaccine efficacy. Inflammasome activation may be important for triggering vaccine-induced humoral and cell-mediated immune responses. Formulations with alum as a typical adjuvant to overcome the effects of host factors have recently been shown to induce inflammasome activation, which augments vaccine efficacy. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is one of the main components of inflammasomes, but it is not clear whether ASC affects the vaccine-induced immune response. Herein, we used two types of vaccines: inactivated influenza vaccine not formulated with alum, and HPV vaccine formulated with alum. We gave the vaccines to ASC knockout (ASC -/- ) mice to investigate the role of ASC in vaccine efficacy. Influenza vaccine-immunized ASC -/- mice did not show antibody titers in week 2 after the first vaccination. After boosting, the antibody titer in ASC -/- mice was about half that in wild type (WT) mice. Furthermore, a cytotoxic T-lymphocyte response against influenza vaccine was not induced in ASC -/- mice. Therefore, vaccinated ASC -/- mice did not show effective protection against viral challenge. ASC -/- mice immunized with alum-formulated HPV vaccine showed similar antibody titers and T-cell proliferation compared with immunized WT mice. However, the HPV vaccine without alum induced up to threefold lower titers of HPV-specific antibody titers in ASC -/- mice compared with those in WT mice. These findings suggest that alum in vaccine can overcome the ASC-deficient condition., (© 2018 The Societies and John Wiley & Sons Australia, Ltd.)

Methylglyoxal (MGO) is a reactive dicarbonyl metabolite of glucose, and its plasma levels are elevated in patients with diabetes. Studies have shown that MGO combines with the amino and sulphhydryl groups of proteins to form stable advanced glycation end products (AGEs), which are associated with vascular endothelial cell (EC) injury and may contribute to the progression of atherosclerosis. In this study, MGO induced apoptosis in a dose-dependent manner in HUVECs, which was attenuated by pre-treatment with z-VAD, a pan caspase inhibitor. Treatment with MGO increased ROS levels, followed by dose-dependent down-regulation of c-FLIP L . In addition, pre-treatment with the ROS scavenger NAC prevented the MGO-induced down-regulation of p65 and c-FLIP L , and the forced expression of c-FLIP L attenuated MGO-mediated apoptosis. Furthermore, MGO-induced apoptotic cell death in endothelium isolated from mouse aortas. Finally, MGO was found to induce apoptosis by down-regulating p65 expression at both the transcriptional and posttranslational levels, and thus, to inhibit c-FLIP L mRNA expression by suppressing NF-κB transcriptional activity. Collectively, this study showed that MGO-induced apoptosis is dependent on c-FLIP L down-regulation via ROS-mediated down-regulation of p65 expression in endothelial cells., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

MicroRNA (miR) can exert various biological functions by targeting oncogenes or tumor suppressor genes in numerous human malignancies. Recent evidence has shown that miR-148a increases the drug sensitivity of various cancer cells. Herein, we show that ectopic expression of miR-148a induces apoptosis, reduces clonogenicity, and increases the sensitivity to TRAIL and cisplatin in renal cancer cells. The luciferase reporter assay showed that miR-148a negatively regulated ras-related protein 14 (Rab14) expression by binding to the miR-148a binding site in the 3' untranslated region (3'UTR) of Rab14. Rab14-specific siRNA-induced downregulation of Rab14 increases the sensitivity to cisplatin, while forced expression of Rab14 lacking 3'-UTR abrogated the pro-apoptotic function of miR-148a in renal cancer cells. These findings suggest that miR-148a acts as a tumor suppressor and holds great potential for renal cancer therapy by directly targeting Rab14.

We reported previously that panaxydol, a component of Panax ginseng roots, induced mitochondria-mediated apoptosis preferentially in transformed cells. This study demonstrates that EGFR activation and the resulting ER stress mediate panaxydol-induced apoptosis, and that panaxydol suppresses in vivo tumor growth in syngeneic and xenogeneic mouse tumor models. In addition, we elucidated that CaMKII and TGF-β-activated kinase (TAK1) participate in p38/JNK activation by elevated cytoplasmic Ca(2+) concentration ([Ca(2+)]c). In MCF-7 cells, EGFR was activated immediately after exposure to panaxydol, and this activation was necessary for induction of apoptosis, suggesting that panaxydol might be a promising anticancer candidate, especially for EGFR-addicted cancer. Activation of PLCγ followed EGFR activation, resulting in Ca(2+) release from the endoplasmic reticulum (ER) via inositol triphosphate and ryanodine receptors. ER Ca(2+) release triggered mitochondrial Ca(2+) uptake indirectly through oxidative stress and ensuing ER stress. Elevated [Ca(2+)]c triggered sequential activation of calmodulin/CaMKII, TAK1 and p38/JNK. As shown previously, p38 and JNK activate NADPH oxidase. Here, it was shown that the resulting oxidative stress triggered ER stress. Among the three signaling branches of the unfolded protein response, protein kinase R-like ER kinase (PERK), but not inositol-requiring enzyme 1 or activating transcription factor 6, played a role in transmitting the apoptosis signal. PERK induced C/EBP homologous protein (CHOP), and CHOP elevated Bim expression, initiating mitochondrial Ca(2+) uptake and apoptosis. In summary, we identified roles of EGFR, the CAMKII-TAK1-p38/JNK pathway, and ER stress in panaxydol-induced apoptosis and demonstrated the in vivo anticancer effect of panaxydol., (© 2015 UICC.)

Dioscin, a saponin extracted from the roots of Polygonatum zanlanscianense, shows several bioactivities such as antitumor, antifungal, and antiviral properties. Although, dioscin is already known to induce cell death in variety cancer cells, the molecular basis for dioscin-induced cell death was not definitely known in cancer cells. In this study, we found that dioscin treatment induced cell death in dose-dependent manner in breast cancer cells such as MDA-MB-231, MDA-MB-453, and T47D cells. Dioscin decreased expressions of Bcl-2 and cIAP-1 proteins, which were down-regulated at the transcriptional level. Conversely, Mcl-1 protein level was down-regulated by facilitating ubiquitin/proteasome-mediated Mcl-1 degradation in dioscin-treated cells. Pretreatment with z-VAD fails to attenuate dioscin-induced cell death as well as caspase-mediated events such as cleavages of procaspase-3 and PARP. In addition, dioscin treatment increased the population of annexin V positive cells and induced DNA fragmentation in a dose-dependent manner in MDA-MB-231 cells. Furthermore, apoptosis inducing factor (AIF) was released from the mitochondria and translocated to the nucleus. Suppression in AIF expression by siRNA reduced dioscin-induced apoptosis in MDA-MB-231 cells. Taken together, our results demonstrate that dioscin-induced cell death was mediated via AIF-facilitating caspase-independent pathway as well as down-regulating anti-apoptotic proteins such as Bcl-2, cIAP-1, and Mcl-1 in breast cancer cells.

One major obstacle in the clinical application of TRAIL as a cancer therapeutic agent is the acquisition of TRAIL resistance. We found that deficiency of TRADD sensitizes cells to TRAIL-induced apoptosis. Enhanced cell death in TRADD(-/-) MEFs is associated with defective NF-κB activation, indicating that the pro-survival function of TRADD in TRAIL signaling is mediated at least in part via NF-κB activation. Moreover, siRNA knock-down of TRADD in cancer cells sensitizes them to TRAIL-induced apoptosis. Thus, TRADD has a survival role in TRAIL signaling and may be one potential target for overcoming TRAIL resistance in cancer therapy., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Panaxydol, a polyacetylenic compound derived from Panax ginseng roots, has been shown to inhibit the growth of cancer cells. In this study, we demonstrated that panaxydol induced apoptosis preferentially in transformed cells with a minimal effect on non-transformed cells. Furthermore, panaxydol was shown to induce apoptosis through an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), activation of JNK and p38 MAPK, and generation of reactive oxygen species (ROS) initially by NADPH oxidase and then by mitochondria. Panaxydol-induced apoptosis was caspase-dependent and occurred through a mitochondrial pathway. ROS generation by NADPH oxidase was critical for panaxydol-induced apoptosis. Mitochondrial ROS production was also required, however, it appeared to be secondary to the ROS generation by NADPH oxidase. Activation of NADPH oxidase was demonstrated by the membrane translocation of regulatory p47(phox) and p67(phox) subunits and shown to be necessary for ROS generation by panaxydol treatment. Panaxydol triggered a rapid and sustained increase of [Ca(2+)](i), which resulted in activation of JNK and p38 MAPK. JNK and p38 MAPK play a key role in activation of NADPH oxidase, since inhibition of their expression or activity abrogated membrane translocation of p47(phox) and p67(phox) subunits and ROS generation. In summary, these data indicate that panaxydol induces apoptosis preferentially in cancer cells, and the signaling mechanisms involve a [Ca(2+)](i) increase, JNK and p38 MAPK activation, and ROS generation through NADPH oxidase and mitochondria.

Berberine (BBR) is an isoquinoline alkaloid which has a wide spectrum of clinical applications including anti-tumor, anti-microbial and anti-inflammatory activities. In this study, we showed that co-treatment with subtoxic doses of BBR and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induced apoptosis in human renal cancer cells, Caki cells, but not in normal tubular kidney cells. Treatment of Caki cells with BBR resulted in downregulation of c-FLIP and Mcl-1 proteins in a dose-dependent manner. The BBR-induced downregulation of c-FLIP and Mcl-1 proteins were involved in proteasome dependent pathways, which was confirmed by the result that pre-treatment with the proteasome inhibitor MG132 inhibited berberine-induced downregulation of the c-FLIP and Mcl-1 proteins. Pretreatment with N-acetyl-L-cysteine (NAC) significantly inhibited the cell death induced by the combined treatment with BBR and TRAIL as well as recovered the expression levels of c-FLIP and Mcl-1 downregulated by treatment with BBR. These results suggested that BBR-stimulated TRAIL-induced apoptosis is dependent on the generation of reactive oxygen species through the downregulation of c-FLIP and Mcl-1 proteins. In conclusion, this study demonstrates that BBR enhances TRAIL-induced apoptosis in human renal cancer cells by ROS-mediated c-FLIP and Mcl-1 down-regulation.

In this study, we identified p53 as a novel TCTP-interacting protein using TCTP as bait. Also, we determined the critical binding sites between TCTP and p53. To elucidate the functional consequence of the interaction, we developed the overexpression and inhibition system of TCTP and p53 expression. Overexpression of TCTP in lung carcinoma cells reversed p53 mediated apoptosis and inhibition of TCTP expression by small interfering RNA increased apoptosis of lung carcinoma cells. Moreover, it was observed that TCTP overexpression promotes degradation of p53. These results clearly indicate that the interaction between TCTP and p53 prevents apoptosis by destabilizing p53. Thus, TCTP acts as a negative regulator of apoptosis in lung cancer., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The role of reactive oxygen species (ROS) in UVB-induced apoptosis has been established, but the molecular mechanisms of their production in response to UVB irradiation in keratinocytes are not well understood. In this study, we demonstrate that levels of BLT2, a low-affinity leukotriene B(4) receptor, and its ligands (LTB(4) and 12(S)-HETE) are greatly increased by UVB irradiation and are responsible for the UVB-induced ROS generation in human keratinocytes. Blockade of BLT2 with a BLT2-specific antagonist, LY255283, or with siBLT2 attenuated ROS production and apoptotic cell death detected by a number of criteria. Moreover, we found that the NADPH oxidase family protein Nox1 lies downstream of BLT2 and mediates UVB-induced ROS production and apoptosis. Topical treatment of mouse epidermal skin with LY255283 gave significant protection against UVB-induced sunburn-associated apoptotic damage. Finally, when BLT2-overexpressing transgenic mice were irradiated with UVB, we observed more extensive skin apoptosis. Taken together, our results demonstrate that a "BLT2-Nox1"-linked pathway has a crucial role in UVB-induced ROS generation and mediates apoptosis in human keratinocytes.

Activating transcription factor 3 (ATF3) is an early-induced gene involved in diverse cellular functions in response to various stresses including viral infection. Here we observed marked reduction of ATF3 by coxsackievirus B3 (CVB3) infection and investigated the regulation and functional role of ATF3 in HeLa cells for the understanding of biological significance of ATF3 downregulation. CVB3 infection markedly reduced ATF3 expression at mRNA and protein levels in parallel with p53 degradation, and preservation of p53 expression rescued CVB3 infection-induced ATF3 downregulation. ATF3 overexpression stimulated apoptotic cell death following CVB3 infection, accompanying with augmentation of CVB3 infection-induced eIF2alpha phosphorylation. However, ATF3 overexpression did not affect viral protein production but promoted virus progeny release. Taken together, our results suggest that ATF3 is under control of p53 in part and that the ATF3 downregulation via p53 degradation may contribute to effective viral production as a modulation mechanism of CVB3 infection-induced cell death.

The effect of surfactin on the proliferation of LoVo cells, a human colon carcinoma cell line, was examined. Surfactin strongly blocked the proliferation of LoVo cells by inducing pro-apoptotic activity and arresting the cell cycle, according to several lines of evidence on DNA fragmentation, Annexin V staining, and altered levels of poly (ADP-ribose) polymerase, caspase-3, p21(WAF1/Cip1), p53, CDK2 and cyclin E. The anti-proliferative activity of surfactin was mediated by inhibiting extracellular-related protein kinase and phosphoinositide 3-kinase/Akt activation, as assessed by phosphorylation levels. Therefore, our data suggest that surfactin may have anti-cancer properties as a result of its ability to downregulate the cell cycle and suppress its survival.

Coxsackievirus B3 (CVB3), an enterovirus in the Picornavirus family, is the most common human pathogen associated with myocarditis and idiopathic dilated cardiomyopathy. We found upregulation of the cysteine-rich protein gene (cyr61) after CVB3 infection in HeLa cells with a cDNA microarray approach, which is confirmed by Northern blot analysis. It is also revealed that the extracellular amount of Cyr61 protein was increased after CVB3 infection in HeLa cells. cyr61 is an early-transcribed gene, and the Cyr61 protein is secreted into the extracellular matrix. Its function is related to cell adhesion, migration, and neuronal cell death. Here, we show that activation of the cyr61 promoter by CVB3 infection is dependent on JNK activation induced by CVB3 replication and viral protein expression in infected cells. To explore the role of Cyr61 protein in infected HeLa cells, we transiently overexpressed cyr61 and infected HeLa cells with CVB3. This increased CVB3 growth in the cells and promoted host cell death by viral infection, whereas down-expression of cyr61 with short interfering RNA reduced CVB3 growth and showed resistance to cell death by CVB3 infection. In conclusion, we have demonstrated a new role for cyr61 in HeLa cells infected with CVB3, which is associated with the cell death induced by virus infection. These data thus expand our understanding of the physiological functions of cyr61 in virus-induced cell death and provide new insights into the cellular factors involved.

Background: Lactucin, a naturally occurring active sesquiterpene lactone, is abundantly found in chicory and romaine lettuce. A recent study reported that lactucin could induce apoptosis in leukemia cells. However, its cytotoxicity and potential molecular mechanisms underlying cancer cell death remain unclear. Objective: Therefore, in this study, we aimed to investigate the direct effect and underlying mechanism of action of lactucin on renal cancer cells. Methods: MTT assay and flow cytometry were performed to evaluate the rate of cell proliferation and apoptosis, respectively. Western blotting, reverse transcription polymerase chain reaction, and protein stability analyses were performed to analyze the effect of lactucin on the expression of apoptosis-related proteins such as B-cell lymphoma 2 (BCL-2) and CFLAR (CASP8 and FADD like apoptosis regulator) long isoform (CFLARL) in Caki-1 human renal cancer cells. In addition, reactive oxygen species (ROS) generation was evaluated using flow cytometry. Results: Lactucin treatment induced apoptosis in Caki-1 cells in a dose-dependent manner via activation of the caspase pathway. It downregulated BCL-2 and CFLARL expression levels by suppressing BCL-2 transcription and CFLARL protein stability, respectively. Pretreatment with N-acetyl-1-cysteine, a ROS scavenger, attenuated the lactucin-induced apoptosis and restored the BCL-2 and CFLARL expression to basal levels. Lactucin-facilitated BCL-2 downregulation was regulated at the transcriptional level through the inactivation of the NF-κB pathway. Conclusions: Our study is the first to demonstrate that lactucin-induced apoptosis is mediated by ROS production, which in turn activates the caspase-dependent apoptotic pathway by inhibiting BCL-2 and CFLARL expression in Caki-1 cells. [ABSTRACT FROM AUTHOR]

Prostate cancer is among the most prevalent malignancies in men. High intake of dairy products is associated with an increased risk of prostate cancer. No study has examined the gene profile changes and molecular mechanism by which casein, milk protein, affects prostate cancer cells. In this study, we used gene expression profiling to identify gene changes in PC3 prostate cancer cells exposed to α-casein. α-casein altered the expression of a large number of genes-related prostate cancer, transcription factor, apoptotic, metabolic, and cell cycle pathways, in addition to the expected cell proliferation signaling pathways. To clarify the molecular events involved in the effect of α-casein on proliferation and progression of PC3 cells, we examined cell proliferation assay, quantitative real-time PCR, Western blotting, and immunohistochemical and immunofluorescence staining. α-casein promoted PC3 cell proliferation and survival under serum-free conditions by increasing the expression of E2F1 and its target gene PCNA. α-casein also protected PC3 cells from serum-starved autophagic cell death by activating the PI3K/Akt pathway through activation of mTORC1, up-regulation of p70S6K, and down-regulation of LC3 autophagosome formation. Our data provide new insights into the molecular mechanisms underlying the tumorigenic effect of α-casein in prostate cancer cells. [ABSTRACT FROM AUTHOR]

Renal cell carcinoma (RCC) is one of the most common types of cancer in adults. Previous studies have reported that the survival rate was significantly lower for renal cancer patients with diabetes than for those without diabetes. Metformin is a well-known anti-diabetic agent used for the treatment of type 2 diabetes mellitus (T2DM). It also inhibits cell proliferation and angiogenesis and is known to possess antitumor effects. However, the molecular mechanism for metformin-induced apoptosis in renal cell carcinoma is not understood. In the present study, treatment with metformin induced apoptosis in A498 cells in a dose-dependent manner. It was revealed that degradation of cellular caspase 8 (FLICE)-like inhibitory protein (c-FLIP) and activation of procaspase-8 were associated with metformin-mediated apoptosis. By contrast, treatment with metformin did not affect the mRNA level of c-FLIPL in A498 cells. Treatment with benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk, a pan-caspase inhibitor) almost completely blocked metformin-induced apoptosis and degradation of c-FLIPL protein. However, N-acetyl-L-cysteine (NAC), a reactive oxygen species (ROS) scavenger, did not inhibit metformin-mediated apoptosis in A498 cells. Taken together, the results of the present study demonstrated that metformin-induced apoptosis involved degradation of the c-FLIPL protein and activation of caspase-8 in human renal cell carcinoma A498 cells and suggested that metformin could be potentially used for the treatment of renal cancer. [ABSTRACT FROM AUTHOR]

Methylglyoxal (MGO) is a reactive dicarbonyl metabolite of glucose, and its plasma levels are elevated in patients with diabetes. Studies have shown that MGO combines with the amino and sulphhydryl groups of proteins to form stable advanced glycation end products (AGEs), which are associated with vascular endothelial cell (EC) injury and may contribute to the progression of atherosclerosis. In this study, MGO induced apoptosis in a dose-dependent manner in HUVECs, which was attenuated by pre-treatment with z-VAD, a pan caspase inhibitor. Treatment with MGO increased ROS levels, followed by dose-dependent down-regulation of c-FLIPL. In addition, pre-treatment with the ROS scavenger NAC prevented the MGO-induced down-regulation of p65 and c-FLIPL, and the forced expression of c-FLIPL attenuated MGO-mediated apoptosis. Furthermore, MGO-induced apoptotic cell death in endothelium isolated from mouse aortas. Finally, MGO was found to induce apoptosis by down-regulating p65 expression at both the transcriptional and posttranslational levels, and thus, to inhibit c-FLIPL mRNA expression by suppressing NF-κB transcriptional activity. Collectively, this study showed that MGO-induced apoptosis is dependent on c-FLIPL down-regulation via ROS-mediated down-regulation of p65 expression in endothelial cells. [ABSTRACT FROM AUTHOR]

Dapagliflozin is a sodium/glucose cotransporter 2 inhibitor used recently to treat patients with type 2 diabetes. A recent study has demonstrated that dapagliflozin induces apoptosis in human renal and breast tumor cells. However, to the best of our knowledge, the molecular mechanism underlying dapagliflozin-mediated apoptosis in Caki-1 human renal carcinoma cells has not been elucidated. The present study demonstrated that the dapagliflozin treatment dose-dependently increased cell death in Caki-1 cells. Dapagliflozin treatment also induced apoptosis as confirmed by FITC-conjugated Annexin V/PI staining. Additionally, treatment with dapagliflozin reduced the expression levels of anti-apoptotic proteins, cellular Fas-associated death domain-like interleukin-1-converting enzyme-inhibitory protein (cFLIP)L and cFLIPS in Caki-1 cells. Benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl-ketone inhibited dapagliflozin-induced apoptosis, implying that dapagliflozin–induced apoptosis is regulated by a caspase-dependent pathway. By contrast, N-acetylcysteine had no effect on dapagliflozin–induced apoptosis and downregulation of cFLIPL and cFLIPS expression. Furthermore, overexpression of cFLIPL, but not cFLIPS, partially inhibited apoptosis induced by dapagliflozin. cFLIPL and cFLIPS mRNA levels remained constant in Caki-1 cells after treatment with 0, 20, 40, 60, 80 and 100 µM dapagliflozin. Notably, it was confirmed that cFLIPS protein levels were reduced due to the increased cFLIPS instability in dapagliflozin-treated Caki-1 cells. The present study also demonstrated that dapagliflozin had no effect on HK-2 normal human kidney cells. Taken together, the present study revealed that dapagliflozin induced apoptosis via the downregulation of cFLIPL and an increase in cFLIPS instability, suggesting that dapagliflozin may be a feasible drug candidate for the treatment of human renal cancer. [ABSTRACT FROM AUTHOR]

Highlights: [•] Methoxychlor (MXC) and triclosan (TCS) increased ovarian cancer cell growth. [•] MXC or TCS resulted in upregulation of cyclin D1 and downregulation of p21 and Bax genes. [•] In addition, they regulated these proteins as well in ovarian cancer cells. [•] MXC and TCS may stimulate ovarian cancer growth via an ER-dependent pathway. [Copyright &y& Elsevier]

Purpose: RH1 is a newly developed bioreductive agent, and its bioactivation is mediated by the enzyme DT-diaphorase (DTD). We have shown previously that RH1 is highly cytotoxic against cells expressing high DTD, using the p53-mutated MDA231 human breast cancer cell line transfected with the DTD gene (D7 cells). We now report that both RH1 and gamma-irradiation cause D7 cells to arrest in the G2/M cell cycle phase and undergo polyploidy. The latter is a way of p53-mutated cells responding to DNA-damaging agents. Only a small proportion of the polyploid cells are clonogenic, hence polyploidy may contribute to the reproductive failure of the cells after RH1 and irradiation. Thus, we investigated the effect of RH1 and gamma-irradiation on the formation of polyploid cells and a sub-G1 population (as a measure of apoptosis) in relation to the G2/M cell cycle block.Methods and Materials: MDA231 D7 cells were treated using a range of RH1 doses. The cells were irradiated using 2 Gy or 5 Gy gamma-rays either as a single dose or in combination with RH1. An IC(90) dose (dose to kill 90% of the cells) of RH1 was administered for 3 h followed by irradiation after a further 24 h. Subsequent changes in cell cycle and polyploidy (DNA content in excess of that of G2/M cells) were examined.Results: Treatment of D7 cells with the RH1 resulted in 60-70% of cells arrested in the G2/M phase of the cell cycle by 24 h, which decreased to control levels by 48 h. Irradiation with 2 Gy and 5 Gy caused a similar G2/M block at 12-24 h, which was followed by a sharp decline at 24-48 h. In contrast, the same dose of radiation combined with RH1 held the cells in the G2/M phase up to 48 h, and this pattern reached pretreatment levels at 72-96 h. Most control cells were found to contain a small number of spontaneously arising polyploid cells. The development of polyploid cells was evident from 12 h after all treatments and showed a significant increase at 48 h and subsequently. As opposed to this, apoptosis measured by the sub-G1 cell population in DNA analyses showed a tendency to increase according to the elapsed time for each group of treatments. Single treatments with RH1 caused a significant increase in the apoptotic population between 48 and 120 h. The first significant increase in apoptosis was observed at 48 h for 5 Gy, 2 Gy + RH1, and 5 Gy + RH1 treatments, and showed a tendency to increase further at later times, but the 2 Gy dose gave an earlier apoptotic peak at 24 h, which decreased to 96 h. The addition of RH1 to the irradiation did not increase the formation of polyploid cells or apoptosis compared with radiation alone (2 Gy vs. RH1 + 2 Gy or 5 Gy vs. RH1 + 5 Gy). The higher dose of irradiation (5 Gy vs. 2 Gy) resulted in a significantly higher proportion of polyploid cells (but not of apoptotic cells) when used alone or in combination (5 Gy + RH1 vs. 2 Gy + RH1).Conclusions: Both RH1 and gamma-irradiation, individually and in combination, showed a significant G2/M block in MDA231 D7 breast cancer cells. The formation of polyploid cells was dependent more on the radiation dose rather than on the pretreatment with RH1. The polyploid cell population was observed after the G2/M cell cycle phase arrest, and it preceded the late increase of the apoptotic cell population. The role of polyploidy in cell reproductive failure in the total cell population is not known, but it appears to contribute to cytotoxicity in cells released from the G2/M cell cycle phase block. [ABSTRACT FROM AUTHOR]

Pioglitazone is an anti-diabetic agent used in the treatment of type 2 diabetes, which belongs to the thiazolidinediones (TZDs) group. TZDs target peroxisome proliferator-activated receptor γ (PPARγ), which functions as a transcription factor of the nuclear hormone receptor. Pioglitazone has antitumor effects in several cancer types and could be a tool for drug therapy in various cancer treatments. Nevertheless, the molecular basis for pioglitazone-induced anticancer effects in renal cancer (RC) has not yet been elucidated. Thus, the aim of the present study was to investigate the detailed signaling pathway underlying pioglitazone-induced apoptosis in Caki cells derived from human clear cell renal cell carcinoma. As a result, it was demonstrated by flow cytometry analysis and Annexin V-propidium iodide staining that pioglitazone treatment induced apoptotic cell death in a dose-dependent manner in Caki cells. The protein expression levels of cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (c-FLIP)(L) and Bcl-2, which were determined by western blotting, decreased after pioglitazone treatment in Caki cells. Flow cytometry and western blot analyses demonstrated that pioglitazone-mediated apoptosis was blocked following pretreatment with the pan-caspase inhibitor, z-VAD-fmk, indicating that pioglitazone-induced apoptosis was mediated via a caspase-dependent signaling pathway. However, the reactive oxygen species (ROS) scavenger, N-acetylcysteine (NAC), did not affect pioglitazone-mediated apoptosis and degradation of c-FLIP(L) and Bcl-2 protein. Of note, it was found by western blot analysis that Bcl-2 protein expression was downregulated by the decreased protein stability of Bcl-2 in pioglitazone-treated Caki cells. In conclusion, these findings indicated that pioglitazone-induced apoptosis is regulated through caspase-mediated degradation of FLIP(L) and reduction of Bcl-2 protein stability, suggesting that pioglitazone is a feasible apoptotic agent that could be used in the treatment of human RC. [ABSTRACT FROM AUTHOR]

Zinc finger with KRAB and SCAN domain 3 (ZKSCAN3) upregulates genes encoding proteins involved in cell differentiation, proliferation and apoptosis. ZKSCAN3 has been reported to be overexpressed in several human cancers such as colorectal cancer and prostate cancer and is proposed as a candidate oncoprotein. However, the molecular mechanism by which ZKSCAN3 participates in carcinogenesis is largely unknown. Here, we evaluated ZKSCAN3 expression in uterine cervical cancers (CC) by immunohistochemistry using formalin-fixed, paraffin-embedded tissues from 126 biopsy samples from 126 patients. The clinicopathological findings were analyzed and compared with ZKSCAN3 expression levels. ZKSCAN3 was strongly overexpressed in CCs compared to adjacent non-neoplastic cervical mucosa tissues. Moreover, a gene copy number assay showed amplified ZKSCAN3 in CC samples. ZKSCAN3 overexpression was also significantly associated with poor overall survival of the patients. Overall, our findings indicate that ZKSCAN3 overexpression is a frequent event in uterine CC and is correlated with a poor clinical outcome. ZKSCAN3 could be developed as a molecular marker for prognostic prediction and early detection. [ABSTRACT FROM AUTHOR]

Abstract: Recently, we found that carbonic anhydrase IX (CA9) modulates tumor-associated cell migration and invasion, and then identified dickkopf-1 (DKK-1) as a novel CA9-interacting protein. In this study, we have determined the binding regions that are required for interaction between CA9 and DKK-1 through in vitro and in vivo. The N-terminal domain of CA9 is participated to interact with the Val60–Tyr168 site of DKK-1. We also observed that DKK-1 inhibits endothelial cell angiogenesis of CA9 in tumorigenesis. Furthermore, induction of CA9-mediated mTOR phosphorylation and angiogenesis was significantly inhibited by over-expression of DKK-1. Taken together, these findings identify DKK-1 as a potential factor in the regulation of CA9 cellular homeostasis and also suggest a new possible role for DKK1-1 in tumorigenesis. [Copyright &y& Elsevier]