The Diarylheptanoid Hirsutenone Sensitizes Chemoresistant Ovarian Cancer Cells to Cisplatin via Modulation of Apoptosis-inducing Factor and X-linked Inhibitor of Apoptosis

Cisplatin (CDDP) and its derivatives are considered first-line treatments for ovarian cancer (OVCA). However, despite initial results that often appear promising, in most cases patients will return with recurrent disease that fails to respond to further chemotherapy. We assayed a number of food phytochemicals with reported PI3K inhibitory ability to identify candidates that can influence CDDP treatment outcomes in chemoresistant OVCA cell lines. A direct comparison revealed that the diarylheptanoid hirsutenone from the tree bark of Alnus hirsuta var. sibirica was superior at inducing CDDP sensitivity in a number of chemoresistant cancer cell lines. Whereas hirsutenone treatment activated p53, its modest efficacy in p53-mutant and -null cell lines suggested the existence of a p53-independent mode of action. Further investigation revealed that hirsutenone causes CDDP-dependent apoptosis in chemoresistant cells by ubiquitin-proteasome-dependent X-linked inhibitor of apoptosis degradation and by enhancing the translocation of apoptosis-inducing factor from the mitochondria to the nucleus. This was found to be, at least in part, under the influence of upstream Akt activity, linking hirsutenone-dependent PI3K inhibition with downstream effects on apoptosis-inducing factor, X-linked inhibitor of apoptosis, and apoptosis. Our findings provide rationale for further investigation of the effects of hirsutenone on chemoresistant OVCA in clinical studies.     

Sensitization by glycerol for CDDP-therapy against human cultured cancer cells and tumors bearing mutated p53 gene

To clarify effective chemotherapeutic regimens against cancer, we examined the effects of glycerol on apoptosis induced by CDDP treatment using cultured human cancer cells (in vitro) and transplanted tumor in mice (in vivo). Human tongue cell carcinoma (SAS) cells transfected with mutated p53 gene (SAS/m p53) showed CDDP-resistance compared with the cells with neo control gene (SAS/ neo). When those cultured cells were pre-treated with glycerol, CDDP-induced apoptosis was enhanced by glycerol in SAS/m p53 cells but not in SAS/ neo cells.In tumor-transplanted mice, the glycerol treatment to tumors enhanced growth delay induced by CDDP in mp53 tumors transplanted with SAS/m p53 cells, but not in wtp53 tumors transplanted with SAS/ neo cells. When transplanted tumors were treated with CDDP alone, the cells positive for active caspase-3, 85 kDa PARP and apoptosis were observed by immunohistochemical staining in wtp53 tumors but not in mp53 tumors. When the tumors were treated with CDDP combined with glycerol, positive cells were observed not only in wtp53 tumors but also in mp53 tumors. These results showed that the CDDP-induced growth inhibition of the tumors is p53 -dependent and that the enhanced growth delay by glycerol may be due to the increased apoptosis. Glycerol might be available for cancer chemotherapy in patients with mp53 tumors.     

Nutlin-3a: A Potential Therapeutic Opportunity for TP53 Wild-Type Ovarian Carcinomas

Epithelial ovarian cancer is a diverse molecular and clinical disease, yet standard treatment is the same for all subtypes. TP53 mutations represent a node of divergence in epithelial ovarian cancer histologic subtypes and may represent a therapeutic opportunity in subtypes expressing wild type, including most low-grade ovarian serous carcinomas, ovarian clear cell carcinomas and ovarian endometrioid carcinomas, which represent approximately 25% of all epithelial ovarian cancer. We therefore sought to investigate Nutlin-3a—a therapeutic which inhibits MDM2, activates wild-type p53, and induces apoptosis—as a therapeutic compound for TP53 wild-type ovarian carcinomas. Fifteen epithelial ovarian cancer cell lines of varying histologic subtypes were treated with Nutlin-3a with determination of IC₅₀ values. Western Blot (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) analyses quantified MDM2, p53, and p21 expression after Nutlin-3a treatment. DNA from 15 cell lines was then sequenced for TP53 mutations in exons 2-11 including intron-exon boundaries. Responses to Nutlin-3a were dependent upon TP53 mutation status. By qRT-PCR and WB, levels of MDM2 and p21 were upregulated in wild-type TP53 sensitive cell lines, and p21 induction was reduced or absent in mutant cell lines. Annexin V assays demonstrated apoptosis in sensitive cell lines treated with Nutlin-3a. Thus, Nutlin-3a could be a potential therapeutic agent for ovarian carcinomas expressing wild-type TP53 and warrants further investigation.     

Vitamin C increases the apoptosis via up-regulation p53 during cisplatin treatment in human colon cancer cells

Vitamin C (VC) is an important antioxidant and enzyme co-factor that works by stimulating the immune system and protecting against infections. It is well known that melanoma cells are more susceptible to VC than any other tumor cells. However, the role of VC in the treatment of colon cancer has not been studied. Cisplatin (CDDP) is a DNA damaging agent and is widely used for treating cancer, while the role of p53 in CDDP-induced cell death has been stressed. Using cell growth assays, morphological methods, Western blotting, flow cytometry, and DNA fragmentation analysis, we measured the expression of p53 level involved in the effect of VC on CDDP-induced apoptosis of HCT116, a human colon cancer cell line. CDDP plus VC treatment resulted in significantly increased apoptosis along with upregulation of p53 compared to untreated cells and/or CDDP-treated cells. These results suggest that VC enhanced CDDP sensitivity and apoptosis via upregulation of p53.     

Apoptosis and chemoresistance in human ovarian cancer: is Xiap a determinant?

Cisplatin-induced apoptosis in epithelial ovarian cancer cells is in part a consequence of suppressed Xiap expression and upregulation of the Fas/FasL system. Changes in the expression of these 'cell death' and 'cell survival' genes lead to activation of caspase-3, and cleavage of MDM2 and FAK. Failure of cancer cells to maintain a balance in the expression of these genes in favor of apoptotic cell death may be an important factor of chemoresistance. Xiap may be a novel target for gene therapy of human ovarian epithelial cancer and, dependent on P53 status, expression of Xiap antisense alone or in combination with wild-type P53 sense may offer a new approach for the treatment of the chemoresistant cancer.     

NSC109268 potentiates cisplatin-induced cell death in a p53-independent manner

Background

Ovarian cancer is the leading cause of death among gynecological cancers. Cisplatin is one of the most effective anticancer drugs used in the treatment of ovarian cancer. Development of resistance to cisplatin limits its therapeutic use. Most of the anticancer drugs, including cisplatin, are believed to kill cancer cells by inducing apoptosis and a defect in apoptotic signaling can contribute to drug resistance. The tumor suppressor protein p53 plays a critical role in DNA damage-induced apoptosis. During a yeast-based drug screening, NSC109268 was identified to enhance cellular sensitivity to cisplatin. The objective of the present study is to determine if p53 is responsible for cisplatin sensitization by NSC109268.

Results

NSC109268 enhanced sensitivity of ovarian cancer 2008 cells and its cisplatin resistant counterpart 2008/C13* cells which express wild-type p53. The potentiation of cisplatin sensitivity by NSC109268 was greater in 2008/C13* cells compared to 2008 cells. Cisplatin caused a concentration-dependent increase in p53 in 2008 and 2008/C13* cells, and the induction of p53 correlated with cisplatin-induced apoptosis as determined by the cleavage of PARP. NSC109268 alone had no effect on p53 but it enhanced p53 level in response to cisplatin. Knockdown of p53 by siRNA, however, did not attenuate cell death in response to cisplatin or combination of NSC109268 and cisplatin.

Conclusions

These results demonstrate that NSC109268 enhances sensitivity of ovarian cancer 2008 cells to cisplatin independent of p53.     

PRIMA-1Met/APR-246 induces wild-type p53-dependent suppression of malignant melanoma tumor growth in 3D culture and in vivo

Disseminating malignant melanoma is a lethal disease highly resistant to radio- and chemotherapy. Therefore, the development of new treatment strategies is strongly needed. Tumor suppressor p53-mediated apoptosis is essential for the response to radio- and chemotherapy. Although p53 is not frequently mutated in melanoma, it is inactivated by integrin αv-mediated signaling, as we previously demonstrated 1, which may account, at least partially, for increased apoptosis resistance of malignant melanoma. In this study we addressed the question whether functional restoration of p53 by APR-246 (PRIMA-1Met), which can reactivate mutant p53 and induce massive apoptosis in cancer cells, is able to restore the function of inactive p53 in melanoma. Using a three-dimensional collagen gel (3D-collagen) to culture melanoma cells carrying wild-type p53, we found that APR-246 treatment resulted in activation of p53, leading to increased expression of p53 pro-apoptotic targets Apaf1 and PUMA and activation of caspase- 9 and -3. Moreover, APR-246 triggered melanoma cell apoptosis that was mediated by p53 and caspase 9. Importantly, APR-246 treatment also suppressed human melanoma xenograft tumors in vivo in a p53-dependent manner. Thus, wild-type p53 reactivation may provide a novel approach for malignant melanoma treatment, with APR-246 as a candidate drug for such a development.     

Piceatannol Enhances Cisplatin Sensitivity in Ovarian Cancer via Modulation of p53, X-linked Inhibitor of Apoptosis Protein (XIAP), and Mitochondrial Fission

Resistance to cisplatin (CDDP) in ovarian cancer (OVCA) arises from the dysregulation of tumor suppressors and survival signals. During genotoxic challenge, these factors can be influenced by secondary agents that facilitate the induction of apoptosis. Piceatannol is a natural metabolite of the stilbene resveratrol found in grapes and is converted from its parent compound by the enzyme CYP1BA1 p450. It has been hypothesized to exert specific effects against various cellular targets; however, its ability to influence CDDP resistance in cancer cells has not been investigated to date. Here, we show that piceatannol is a potent enhancer of CDDP sensitivity in OVCA, and this effect is achieved through the modulation of several major determinants of chemoresistance. Piceatannol enhances p53-mediated expression of the pro-apoptotic protein NOXA, increases XIAP degradation via the ubiquitin-proteasome pathway, and enhances caspase-3 activation. This response is associated with an increase in Drp1-dependent mitochondrial fission, leading to more effective induction of apoptosis. In vivo studies using a mouse model of OVCA reveal that a number of these changes occur in association with a greater overall reduction in tumor weight when mice are treated with both piceatannol and CDDP, in comparison to treatment with either agent alone. Taken together, these findings demonstrate the potential application of piceatannol to enhance CDDP sensitivity in OVCA, and it acts on p53, XIAP, and mitochondrial fission.     

Transfection of mutant p53 gene depresses X-ray- or CDDP-induced apoptosis in a human squamous cell carcinoma of the head and neck

The present study examined whether X-ray- and CDDP-sensitivities depend on p53 gene status in human squamous cell carcinoma of the head and neck (SAS cells) showing dominant negative nature of mutant p53 protein. SAS cells were transfected with a vector carrying a mutant p53 gene (SAS/Trp248 cells) or neomycin resistant gene control vector (SAS/neo cells). Sensitivities of the transfected cells to X-ray or CDDP were measured with colony formation assay. The incidence of apoptosis by X-ray or CDDP was analyzed with Hoechst staining or DNA ladder formation assay. The activation of caspase-3 was estimated as an indicator of apoptosis by the detection of fragmentation of caspase-3 or poly (ADP ribose) polymerase (PARP) with Western blot. SAS/Trp248 cells showed X-ray- and CDDP-resistance due to the dominant negative nature of mutant p53, compared with SAS/neo cells. The incidence of DNA ladders and apoptotic bodies increased markedly in SAS/neo cells after X-ray irradiation or CDDP treatment, but increased only slightly in SAS/Trp248 cells. Fragmentation of caspase-3 and PARP was observed in SAS/neo cells, but almost no such fragmentation was observed in SAS/Trp248 cells after X-ray irradiation or CDDP treatment. The present results strongly suggest that the X-ray- and CDDP-sensitivities of human squamous cell carcinomas are p53-dependent, and that the sensitivities are tightly correlated with the induction of apoptosis through caspase-3 activation. The p53-dependent X-ray- or CDDP-sensitivity was supported by results from p53-null human lung cancer H1299 cells which were transfected with wild-type or mutant p53 gene.     

Testis developmental related gene 1 regulates the chemosensitivity of seminoma TCam‐2 cells to cisplatin via autophagy

We previously identified testis developmental related gene 1 (TDRG1), a gene implicated in proliferation of TCam‐2 seminoma cells. Recent evidence has revealed that autophagy influences the chemosensitivity of cancer cells to chemotherapy. However, whether TDRG1 protein regulates autophagy in seminoma cells and influences their sensitivity to cis‐dichlorodiammine platinum (CDDP) remains unknown. In this study, we used TCam‐2 cells and male athymic BALB/c nude mice with xenografts of TCam‐2 cells to investigate autophagy, cell viability, apoptosis and the p110β/Rab5/Vps34 (PI3‐kinase Class III) pathway under the conditions of TDRG1 overexpression or knockdown and with or without CDDP treatment. We found that TDRG1 upregulation promoted autophagy in both TCam‐2 cells and seminoma xenografts via p110β/Rab5/Vps34 activation. Inhibition of autophagy reduced cell viability and promoted apoptosis during CDDP treatment of TCam‐2 cells. Similarly, TDRG1 knockdown inhibited autophagy, reduced cell viability and promoted apoptosis during CDDP treatment of TCam‐2 cells. TDRG1 knockdown inhibited tumour growth and promoted apoptosis in TCam‐2 cell xenografts, whereas TDRG1 overexpression had the opposite effect. According to these results, we propose that high expression of TDRG1 promotes autophagy through the p110β/Rab5/Vps34 pathway in TCam‐2 cells. TDRG1 overexpression promotes autophagy and leads to CDDP resistance, whereas TDRG1 knockdown inhibits autophagy and promotes chemosensitivity to CDDP both in vivo and in vitro. This study has uncovered a novel role of TDRG1 in reducing chemoresistance during CDDP treatment and provides potential therapeutic strategies for the treatment of human seminoma.     

Trichostatin A sensitizes HBx-expressing liver cancer cells to etoposide treatment

Agents commonly used in cancer chemotherapy rely on the induction of cell death via apoptosis, mitotic catastrophe, premature senescence and autophagy. Chemoresistance is the major factor limiting long-term treatment success in patients with hepatocellular carcinoma (HCC). Recent studies have revealed that the hepatitis B virus X protein (HBx) exerts anti-apoptotic effects, resulting in an increased drug resistance in HCC cells. In this study, we showed that etoposide treatment activated caspase-8 and caspase-3, leading to cleavages of p53, Bid and PARP, which subsequently induced apoptosis. Furthermore, p53 and Bid were accumulated in cytoplasm following etoposide treatment. However, HBx significantly attenuated etoposide-induced cell death. In HBx-expressing cells, despite the translocation of p53 and Bid to cytoplasm, the activation of caspases was inhibited. Furthermore, the phosphorylation of extracellular-signal-regulated kinase (ERK) was markedly increased in HBx-expressing cells. Moreover, the pretreatment with trichostatin A (TSA, a histone deacetylase inhibitor) or TSA in combination with etoposide significantly sensitized HCC cells to apoptosis by inhibiting ERK phosphorylation, reactivating caspases and PARP, and inducing translocation of p53 and Bid to cytoplasm. Collectively, HBx reduces the sensitivity of HCC cells to chemotherapy. TSA in combination with etoposide can significantly overcome the increased resistance of HBx-expressing HCC cells to chemotherapy.     

Recombinant GnRH-p53 protein sensitizes breast cancer cells to 5-fluorouracil-induced apoptosis in vitro and in vivo

An ideal approach to treat cancers with dysfunctional p53 tumor suppressor gene is to reinstate p53 functionality by directly using p53 protein as a therapeutic agent. However, this has not been possible because the cells cannot readily internalize the protein. We constructed a fusion protein consisting of gonadotropin-releasing hormone (GnRH-p53) and p53 moieties. The recombinant protein was directly used to treat human breast cancer cells and athymic nude mice bearing breast cancer xenografts, with or without DNA synthesis-arresting agent 5-fluorouracil (5-FU). Treatments of cells from breast cancer cell-lines MDA-MB-231, T47D, or SKBR-3 with GnRH-p53 in combination with 5-FU significantly enhanced p53-activated apoptosis signals, including PUMA expression, BAX translocation to mitochondria, and activated caspase-3. Intratumoral injection of the GnRH-p53 protein inhibited MDA-MB-231 xenograft growth and induced p53-mediated apoptosis in the tumors. Systemic treatment of the tumor-bearing mice via tail vein injection of GnRH-p53 markedly augmented the anticancer efficacy of 5-FU. Substitution of GnRH-p53 with wild type p53 protein had no effect. Recombinant GnRH-p53 is able to function as a surrogate of p53 with regard to its apoptosis-inducing activity. Combination of GnRH-p53 with DNA-damaging drugs may be of important therapeutic value for cancer treatment.     

Disruption of the MDM2�Cp53 interaction strongly potentiates p53-dependent apoptosis in cisplatin-resistant human testicular carcinoma cells via the Fas/FasL pathway

Wild-type p53 has a major role in the response and execution of apoptosis after chemotherapy in many cancers. Although high levels of wild-type p53 and hardly any TP53 mutations are found in testicular cancer (TC), chemotherapy resistance is still observed in a significant subgroup of TC patients. In the present study, we demonstrate that p53 resides in a complex with MDM2 at higher cisplatin concentrations in cisplatin-resistant human TC cells compared with cisplatin-sensitive TC cells. Inhibition of the MDM2–p53 interaction using either Nutlin-3 or MDM2 RNA interference resulted in hyperactivation of the p53 pathway and a strong induction of apoptosis in cisplatin-sensitive and -resistant TC cells. Suppression of wild-type p53 induced resistance to Nutlin-3 in TC cells, demonstrating the key role of p53 for Nutlin-3 sensitivity. More specifically, our results indicate that p53-dependent induction of Fas membrane expression (∼threefold) and enhanced Fas/FasL interactions at the cell surface are important mechanisms of Nutlin-3-induced apoptosis in TC cells. Importantly, an analogous Fas-dependent mechanism of apoptosis upon Nutlin-3 treatment is executed in wild-type p53 expressing Hodgkin lymphoma and acute myeloid leukaemia cell lines. Finally, we demonstrate that Nutlin-3 strongly augmented cisplatin-induced apoptosis and cell kill via the Fas death receptor pathway. This effect is most pronounced in cisplatin-resistant TC cells.     

Aciculatin Induces p53-Dependent Apoptosis via MDM2 Depletion in Human Cancer Cells In Vitro and In Vivo

Aciculatin, a natural compound extracted from the medicinal herb Chrysopogon aciculatus, shows potent anti-cancer potency. This study is the first to prove that aciculatin induces cell death in human cancer cells and HCT116 mouse xenografts due to G1 arrest and subsequent apoptosis. The primary reason for cell cycle arrest and cell death was p53 accumulation followed by increased p21 level, dephosphorylation of Rb protein, PUMA expression, and induction of apoptotic signals such as cleavage of caspase-9, caspase-3, and PARP. We demonstrated that p53 allele-null (-/-) (p53-KO) HCT116 cells were more resistant to aciculatin than cells with wild-type p53 (+/+). The same result was achieved by knocking down p53 with siRNA in p53 wild-type cells, indicating that p53 plays a crucial role in aciculatin-induced apoptosis. Although DNA damage is the most common event leading to p53 activation, we found only weak evidence of DNA damage after aciculatin treatment. Interestingly, the aciculatin-induced downregulation of MDM2, an important negative regulator of p53, contributed to p53 accumulation. The anti-cancer activity and importance of p53 after aciculatin treatment were also confirmed in the HCT116 xenograft models. Collectively, these results indicate that aciculatin treatment induces cell cycle arrest and apoptosis via inhibition of MDM2 expression, thereby inducing p53 accumulation without significant DNA damage and genome toxicity.     

The anti-tumor effect of cross-reacting material 197, an inhibitor of heparin-binding EGF-like growth factor, in human resistant ovarian cancer

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a promising target for ovarian cancer therapy. Cross-reacting material 197 (CRM197), a specific HB-EGF inhibitor, has been proven to represent possible chemotherapeutic agent for ovarian cancer. However, the effect of CRM197 on the resistant ovarian carcinoma cells has not been sufficiently elucidated. Here, we found that HB-EGF was over-expressed in a paclitaxel-resistant human ovarian carcinoma cell line (A2780/Taxol) and a cisplatin-resistant cell line (A2780/CDDP), as well as the xenograft mouse tissue samples with these cells. To investigate the possible significance of the HB-EGF over-expression in A2780/Taxol and A2780/CDDP cells, we inhibited HB-EGF expression by CRM197 to investigate the effect of CRM197 treatment on these cells. We observed that CRM197 significantly induced anti-proliferative activity in a dose-dependent manner with the cell-cycle arrest at the G0/G1 phase and enhanced apoptosis in A2780/Taxol and A2780/CDDP cells. The sensitive ovarian carcinoma parental cell line (A2780), A2780/Taxol and A2780/CDDP cells formed tumors in nude mice, and enhanced tumorigenicity was observed in drug-resistant tumors. Furthermore, we observed that CRM197 significantly suppressed the growth of drug-resistant ovarian cancer xenografts in vivo (p<0.001). These results suggest that CRM197 as an HB-EGF-targeted agent has potent anti-tumor activity in paclitaxel- and cisplatin-resistant ovarian cancer which over-express HB-EGF.