Cisplatin-induced Apoptotic Cell Death Research Articles

Phospholipase C-related catalytically inactive protein enhances cisplatin-induced apoptotic cell death

Cisplatin is one of the most widely used chemotherapeutic agents and induces caspase-9-mediated apoptosis. Here, we examined whether phospholipase C-related catalytically inactive protein (PRIP) enhances cisplatin-induced apoptosis of breast cancer cells. PRIP depletion increased expression of X-linked inhibitor of apoptosis protein (XIAP) by inhibiting protein degradation, which is downstream of the phosphatidylinositol 3-kinase/AKT pathway and inhibits apoptotic signaling by blocking caspase-9 activation. Conversely, the viability of MCF-7 cells transfected with Prip1 was significantly lower than that of control cells in the presence of cisplatin. The number of apoptotic nuclei and expression levels of cleaved caspase-9 and downstream cleaved caspase-7 and poly-ADP ribose polymerase were greater in PRIP1-expressing MCF-7 cells treated with cisplatin than in control cells. XIAP was decreased by expression of pleckstrin homology domain of PRIP1 (PRIP1-PH domain) that blocked phosphatidylinositol 4,5 bisphosphate metabolism. In an orthotopic transplantation model, combined administration of PRIP1-PH domain-containing liposomes and cisplatin reduced the size of MCF-7 tumors compared with cisplatin alone. Our findings demonstrate that PRIP promotes XIAP degradation by inhibiting PI(3,4,5)P3/AKT signaling and enhances cisplatin-induced apoptotic cell death. Therefore, we propose that PRIP1-PH liposomes are a novel agent to avoid cisplatin resistance.

Benzyl salicylate from the stems and stem barks of Cornus walteri as a nephroprotective agent against cisplatin-induced apoptotic cell death in LLC-PK1 cells.

Nephrotoxicity currently plagues the therapeutic use of cisplatin. Its major side effects are predominantly associated with renal tubular cell apoptosis. In this study, we examined the potential nephroprotective constituents from C. walteri that can exert effects against cisplatin-induced oxidative stress and apoptosis in renal tubular epithelial (LLC-PK1) cells. Phytochemical investigation of the MeOH extract of the stems and stem barks of C. walteri led to the isolation of the potential nephroprotective constituents (1–15). Among the isolates, treatment with benzyl salicylate (BS) (15) (50 and 100 μM) significantly attenuated LLC-PK1 cell apoptosis and potently blocked cisplatin-induced nuclei condensation, intracellular ROS generation, and apoptosis, as indicated by Hoechst 33342 and H2DCFDA staining. Furthermore, the expression of apoptosis-related proteins such as MAPK, Bax/Bcl-2 protein ratio, and cleaved caspase-9 and -3, were significantly decreased by BS. These findings provide that benzyl salicylate can be a nephroprotective agent against cisplatin-induced oxidative stress and apoptosis.

Protective Effect of Panaxynol Isolated from Panax vietnamensis against Cisplatin-Induced Renal Damage: In Vitro and In Vivo Studies.

Polyacetylenic compounds isolated from Panax species are comprised of non-polar C17 compounds, exhibiting anti-inflammatory, antitumor, and antifungal activities. Panaxynol represents the major component of the essential oils of ginseng. We investigated whether panaxynol isolated from Panax vietnamensis (Vietnamese ginseng, VG) could prevent cisplatin-induced renal damage induced in vitro and in vivo. Cisplatin-induced apoptotic cell death was observed by staining with annexin V conjugated with Alexa Fluor 488, and western blotting evaluated the molecular mechanism. Panaxynol at concentrations above 0.25 μM prevented cisplatin-induced LLC-PK1 porcine renal proximal tubular cell death. LLC-PK1 cells treated with cisplatin demonstrated an increase in apoptotic cell death, whereas pretreatment with 2 and 4 μM panaxynol decreased this effect. Cisplatin demonstrated a marked increase in the phosphorylation of c-Jun N-terminal kinase (JNK), P38, and cleaved caspase-3. However, pretreatment with 2 and 4 μM panaxynol reversed the upregulated phosphorylation of JNK, P38, and the expression of cleaved caspase-3. We confirmed that the protective effect of panaxynol isolated from P. vietnamensis in LLC-PK1 cells was at least partially mediated by reducing the cisplatin-induced apoptotic damage. In the animal study, panaxynol treatment ameliorated body weight loss and blood renal function markers and downregulated the mRNA expression of inflammatory mediators.

Abstract 4745: Reversal by inhibiting XBP1 activation of glucose deprivation-induced tolerance to multiple anticancer therapies

Abstract Tumor microenvironment is harsh condition including low pH, hypoxia and nutrient deprivation. Cancer cells can switch their phenotypes and characteristics dependent on the microenvironment. Most previous researches have focused mainly on the effect of hypoxia on cancer cells plasticity and resistance to anticancer therapies. Despite the accumulation of emerging evidences, the effect of nutrient deficiency to multiple anticancer therapies is still unclear.In this study, human lung adenocarcinoma cells (A549), triple-negative breast cancer (MDA-MB-231), hepatocellular carcinoma cells (HepG2), pancreas adenocarcinoma cells (AsPC-1) were cultured in the medium containing low concentrations of glucose. Low sensitivity to cisplatin or taxol was observed in the cancer cells maintained for 28 weeks or so under the glucose-deprived condition. In addition, the multidrug tolerant cells were also insensitive to cytotoxic T cell (CTL)-based immunotherapy and had increased migration ability compared with control group under normal glucose condition. Reversion to normal glucose condition only for short term period (at least 4 days) made the multidrug tolerant cells susceptible to cisplatin-induced apoptotic cell death. In the unfolded protein response, BiP-XBP1 signaling was increased but PERK signaling was decreased in response to long-term glucose deprivation. Sensitivity to cisplatin treatment and CTL-based immunotherapy was restored by inhibiting XBP1 splicing in glucose deprivation-induced multidrug tolerant cells in various types of cancer.This study demonstrated that glucose deprivation-induced tolerance to multiple anti-cancer therapies is transient and reversible, and requires activation of IRE1-XBP1 pathway.This research was supported by the National Research Foundation of Korea (2018R1D1A1B07045297, 2016R1D1A1A02936945 and 2017R1D1A1B03036008). Citation Format: Mini Jeong, Serin Jo, Serkin Park, Jeongwon Sohn, Yun Gyu Park. Reversal by inhibiting XBP1 activation of glucose deprivation-induced tolerance to multiple anticancer therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4745.

Novel Atg4B inhibitors potentiate cisplatin therapy in lung cancer cells through blockade of autophagy

Autophagy induction in tumors is implicated in tumor malignancy and acquisition of anticancer-drug resistance, and hence, autophagy inhibitors are expected as novel anticancer agents. Atg4B, a cysteine proteinase, is a key enzyme in autophagy process, and considered as one of desirable targets for development of selective autophagy inhibitors. In this study, to search novel inhibitors of Atg4B, we employed the virtual screening approach and differential scanning fluorimetry analysis, and found two compounds 1 (2-methyl-5-oxo-5H-[1,3,4]thiadiazolo-[3,2-a]pyrimidine-6-carboxylic acid) and 17 (methyl 4-({2-[(4,6-diaminopyrimidin-2-yl)sulfanyl]propanoyl} amino)benzoate), showing IC50 values of 10 and 12 µM, respectively, in in vitro LC3 cleavage assay of Atg4B. Molecular docking analysis suggested that 1 and 17 bind to the catalytic domain of Atg4B. The treatments with 1 and 17 significantly suppressed autophagy induction to lung cancer A549 cells by tamoxifen and cisplatin, in which their cotreatments promoted tamoxifen- and cisplatin-induced apoptotic cell death. Moreover, the autophagy suppression by 1 and 17 remarkably restored the sensitivity to cisplatin in cisplatin-resistant A549 cells. Thus, 1 and 17 represent promising leads for future development of potent and selective Atg4B inhibitors that augment existing chemotherapy efficacy.

Blockade of ALK4/5 signaling suppresses cadmium- and erastin-induced cell death in renal proximal tubular epithelial cells via distinct signaling mechanisms.

Various types of cell death, including apoptosis, necrosis, necroptosis, and ferroptosis, are induced in renal tubular epithelial cells following exposure to environmental stresses and toxicants such as osmotic stress, ischemia/reperfusion injury, cisplatin, and cadmium. This is known to cause renal dysfunction, but the cellular events preceding stress-induced cell death in renal tubules are not fully elucidated. The activin receptor-like kinase (ALK) 4/5, also known as activin-transforming growth factor (TGF) β receptor, is involved in stress-induced renal injury. We, therefore, studied the role of ALK4/5 signaling in HK-2 human proximal tubular epithelial cell death induced by cisplatin, cadmium, hyperosmotic stress inducer, sorbitol, and the ferroptosis activator, erastin. We found that ALK4/5 signaling is involved in cadmium- and erastin-induced cell death, but not sorbitol- or cisplatin-induced apoptotic cell death. Cadmium exposure elevated the level of phosphorylated Smad3, and treatment with the ALK4/5 kinase inhibitors, SB431542 or SB505124, suppressed cadmium-induced HK-2 cell death. Cadmium-induced cell death was attenuated by siRNA-mediated ALK4 or Smad3 silencing, or by treatment with SIS3, a selective inhibitor of TGFβ1-dependent Smad3 phosphorylation. Furthermore, ALK4/5 signaling activated Akt signaling to promote cadmium-induced HK-2 cell death. In contrast, siRNA-mediated Inhibin-bA silencing or treatment with TGFβ1 or activin A had little effect on cadmium-induced HK-2 cell death. On the other hand, treatment with SB431542 or SB505124 attenuated erastin-induced ferroptosis by hyperactivating Nrf2 signaling in HK-2 cells. These results suggest that blockade of ALK4/5 signaling protects against cadmium- and erastin-induced HK-2 cell death via Akt and Nrf2 signaling pathways, respectively.

Inhibition of autophagy promotes cisplatin-induced apoptotic cell death through Atg5 and Beclin 1 in A549 human lung cancer cells.

Recent studies have indicated that autophagy contributes to tumorigenesis and participates in acquired chemotherapeutic resistance. The present study aimed to determine the function and underlying mechanism of cisplatin‑induced autophagy in A549 human lung cancer cells. Autophagy was measured by LC3B‑I/II conversion, LC3B puncta and autophagosomes formation. Apoptotic cell death was measured by caspase‑3 activity, caspase‑3 cleavage and LDH release. The transcriptional and expressional level of autophagy related proteins were measured by reverse transcription-quantitative polymerase chain reaction and western blot analysis. Beclin 1 and Atg 5 siRNA transfection was used to explore the function of cisplatin‑induced autophagy. The results demonstrated that cisplatin induces apoptotic cell death in A549 cells and triggers an autophagic response, as indicated by increased microtubule‑associated protein1 light chain 3β (LC3B)‑I/II conversion, increased LC3B puncta and autophagosome formation. Mechanisms underlying cisplatin‑induced autophagic responses were also investigated. Cisplatin induced autophagy by upregulating the mRNA and protein expression levels of autophagy protein (Atg)5 and Beclin 1, whereas the mRNA and protein expression levels of serine/threonine‑protein kinase ULK1, Atg3, Atg7, Atg12, and sequestosome‑1 were not markedly upregulated. In addition, knockdown of Atg5 and Beclin 1 by small interfering RNA transfection impaired cisplatin‑induced activation of autophagic responses, increased caspase‑3 cleavage and inhibited cell viability. These findings suggested that disruption of autophagy via the inhibition of Atg5 and Beclin 1 may promote cisplatin‑induced apoptotic cell death in A549 human lung cancer cells. In conclusion, the present study demonstrated that targeting autophagy may be used in the future for the treatment of lung cancer.

Adipose-derived stem cell conditioned medium attenuates cisplatin-triggered apoptosis in tongue squamous cell carcinoma.

Autologous fat grafting procedures have noted a markedly increased frequency, not only for cosmetic purposes, but also for deformities after head and neck cancer and breast cancer surgery. Carcinogenesis is always a major concern in cell therapy-related issues. However, there is no literature discussing this issue in head and neck squamous cell carcinoma patients. To evaluate the interaction of tongue cancer cells and adipose-derived stem cells, we performed a series of invitro experiments. Our results demonstrated that cisplatin significantly reduced the viabilities of SCC‑25 and CAL‑27 cells in a concentration-dependent manner, but it had low cytotoxicity in cisplatin-resistant CAL‑27 (CAR) cells. There was no significant difference in terms of viability among the SCC‑25, CAL‑27, and CAR cells in the adipose-derived stem cell conditioned medium and control groups. There was also no significant difference in terms of cell migration as determined by wound healing assay of SCC‑25, CAL‑27, and CAR cells between the adipose-derived stem cell conditioned medium treatment and control treatment. Importantly, the adipose-derived stem cell conditioned medium attenuated cisplatin-triggered cell death in the SCC‑25 and CAL‑27 cells. Moreover, adipose-derived stem cell conditioned medium markedly inhibited cisplatin-induced apoptotic cell death (sub‑G1 phase) in the CAL‑27 cells. Western blot analyses indicated that cisplatin-induced reductions in pro‑caspase‑3, pro‑caspase‑9, phospho-BAD, phospho-IGF-1R, phospho-AKT, and phospho-ERK in CAL‑27 cells were reversed by adipose-derived stem cell conditioned medium supplement. Taken together, we provide evidence that adipose-derived stem cell conditioned medium protects CAL‑27 cells from cisplatin-induced cell death, possibly through upregulation of the IGF-1R/AKT/ERK signaling pathway.

Protective Effects of Gemigliptin, a Dipeptidyl Peptidase-4 Inhibitor, against Cisplatin-Induced Nephrotoxicity in Mice.

Dipeptidyl peptidase-4 (DPP-4) inhibitors are widely used antihyperglycemic agents for the treatment of type 2 diabetes mellitus. Recently, the pleiotropic actions of DPP-4 inhibitors have drawn much attention. In the present study, we aimed to examine whether gemigliptin, a recently developed DPP-4 inhibitor, could protect against cisplatin-induced nephrotoxicity. We showed that pretreatment with gemigliptin attenuated cisplatin-induced renal dysfunction, as shown by analysis of plasma creatinine levels and blood urea nitrogen and histological damage. Elevated plasma levels of active glucagon-like peptide-1 were observed in gemigliptin-pretreated mice after cisplatin treatment, compared to that in cisplatin alone-treated mice. Gemigliptin attenuated cisplatin-induced apoptotic cell death, as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and Western blot analysis in the kidneys. Gemigliptin also decreased the plasma levels of tumor necrosis factor-α and monocyte chemoattractant protein-1 and attenuated nuclear staining of nuclear factor kappa-B p65 in the kidneys. In addition, gemigliptin increased the protein expression of heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1) in the kidneys of cisplatin-treated mice. Taken together, these results suggest that pretreatment with gemigliptin protects against cisplatin-induced nephrotoxicity in mice, possibly via inhibition of apoptotic cell death and inflammatory responses through induction of HO-1 and NQO1 expression.

GFRA1 promotes cisplatin-induced chemoresistance in osteosarcoma by inducing autophagy

ABSTRACTRecent progress in chemotherapy has significantly increased its efficacy, yet the development of chemoresistance remains a major drawback. In this study, we show that GFRA1/GFRα1 (GDNF family receptor α 1), contributes to cisplatin-induced chemoresistance by regulating autophagy in osteosarcoma. We demonstrate that cisplatin treatment induced GFRA1 expression in human osteosarcoma cells. Induction of GFRA1 expression reduced cisplatin-induced apoptotic cell death and it significantly increased osteosarcoma cell survival via autophagy. GFRA1 regulates AMPK-dependent autophagy by promoting SRC phosphorylation independent of proto-oncogene RET kinase. Cisplatin-resistant osteosarcoma cells showed NFKB1/NFκB-mediated GFRA1 expression. GFRA1 expression promoted tumor formation and growth in mouse xenograft models and inhibition of autophagy in a GFRA1-expressing xenograft mouse model during cisplatin treatment effectively reduced tumor growth and increased survival. In cisplatin-treated patients, treatment period and metastatic status were associated with GFRA1-mediated autophagy. These findings suggest that GFRA1-mediated autophagy is a promising novel target for overcoming cisplatin resistance in osteosarcoma.

Intracellular prostaglandin E2 mediates cisplatin-induced proximal tubular cell death

Nephrotoxicity, particularly in the proximal tubule, limits the therapeutic efficacy of the antineoplastic drug cisplatin. The signaling mechanisms appear to be multifactorial, involving inflammation, oxidative stress, and caspase. Here we studied the role of intracellular prostaglandin E2 (iPGE2) in cisplatin's cytotoxicity in human proximal tubular HK-2 cells. Cisplatin-induced apoptotic cell death was prevented by inhibitors of the prostaglandin transporter (PGT) or by PGT knock-down or by pharmacologic inhibition of PGE2 EP receptors or cyclo-oxygenase-2 (COX-2). iPGE2 also increased in cisplatin-treated cells, which was probably due to increased expression of COX-2, microsomal PGE2 synthase-1 and PGT, and was prevented by inhibitors of PGT or COX-2. Thus iPGE2, most likely acting through intracellular EP receptors, mediates cisplatin-induced HK-2 cell death. Importantly, the tumoricidal effect of cisplatin on human cervical adenocarcinoma HeLa cells was not affected by a pharmacologic inhibitor of PGT. In conclusion, iPGE2 may play a significant role in the pathogenesis of cisplatin’s nephrotoxicity and treatment with PGT inhibitors might represent a novel strategy in its prevention.

Vaccine-induced tumor necrosis factor-producing T cells synergize with cisplatin to promote tumor cell death.

Cancer immunotherapy, such as vaccination, is an increasingly successful treatment modality, but its interaction with chemotherapy remains largely undefined. Therefore, we explored the mechanism of synergy between vaccination with synthetic long peptides (SLP) of human papillomavirus type 16 (HPV16) and cisplatin in a preclinical tumor model for HPV16. SLP vaccination in this preclinical tumor model allowed the elucidation of novel mechanisms of synergy between chemo- and immunotherapy. By analyzing the tumor immune infiltrate, we focused on the local intratumoral effects of chemotherapy, vaccination, or the combination. Of several chemotherapeutic agents, cisplatin synergized best with SLP vaccination in tumor eradication, without requirement for the maximum-tolerated dose (MTD). Upon SLP vaccination, tumors were highly infiltrated with HPV-specific, tumor necrosis factor-α (TNFα)- and interferon-γ (IFNγ)-producing T cells. Upon combined treatment, tumor cell proliferation was significantly decreased compared with single treated and untreated tumors. Furthermore, we showed that TNFα strongly enhanced cisplatin-induced apoptotic tumor cell death in a JNK-dependent manner. This is consistent with upregulation of proapoptotic molecules and with enhanced cell death in vivo upon combined SLP vaccination and cisplatin treatment. In vivo neutralization of TNFα significantly reduced the antitumor responses induced by the combined treatment. Taken together, our data show that peptide vaccination with cisplatin treatment leads to decreased tumor cell proliferation and TNFα-induced enhanced cisplatin-mediated killing of tumor cells, together resulting in superior tumor eradication.

Protective role of quercetin against cisplatin-induced hair cell damage in zebrafish embryos.

The aim of this study was to evaluate the protective effects of quercetin on cisplatin-induced hair cell damage in transgenic zebrafish embryos. Five days postfertilization zebrafish embryos were exposed to 1 mM cisplatin and quercetin at 10, 50, 100, or 200 μM for 4 h. Hair cells within neuromasts of the supraorbital, otic, and occipital lateral lines were analyzed by fluorescent microscopy (n = 10). Survival of hair cells was calculated as the average number of hair cells in the control group that were not exposed to cisplatin. Ultrastructural changes were evaluated using scanning electron microscopy. Hair cell damage in neuromasts was decreased by co-treatment of quercetin and cisplatin (quercetin 100 μM: 8.6 ± 1.1 cells; 1 mM cisplatin only: 5.0 ± 0.5 cells; n = 10, p < 0.05); apoptosis of hair cells examined by special stain was also decreased by quercetin. The ultrastructure of hair cells within neuromasts was preserved in zebrafish by the combination of quercetin (100 μM) and cisplatin (1 mM). In conclusion, quercetin showed protective effects against cisplatin-induced toxicity in a zebrafish model. The results of this study suggest the possibility of a protective role of quercetin against cisplatin-induced apoptotic cell death in zebrafish.

ATG7 deficiency promote apoptotic death induced by Cisplatin in human esophageal squamous cell carcinoma cells

Cisplatin-(DDP)-based adjuvant chemotherapy is widely used for the treatment of esophageal cancer. However, DDP-based combinatorial treatments can eventually result in tumor resistance response. Therefore, new therapeutic strategies and/or new adjuvant drugs still need to be explored. In this study, we aimed to understand the role of autophagy in ESCC cells resistance to Cisplatin and discuss its potential therapeutic implication. We found that exposure to Cisplatin induced a significant increase in LC3 formation. While the proliferation of ESCC cells was inhibited upon Cisplatin exposure, inhibition of autophagy by ATG7 interference further increased the sensitivity to chemotherapy. Meanwhile, the Cisplatin-induced apoptotic cell death was significantly enhanced. These results suggest that autophagy may function importantly in ESCC cells resistance to Cisplatin. Intriguingly, the resistance could be recovered by autophagy inhibition. This also points to potential therapy for ESCC by perturbing autophagy.

Abstract 894: Melanoma fights cisplatin with NO: S-nitrosation as a mechanism supporting drug-resistance.

Abstract Melanoma patients experience inferior survival after bio-chemotherapy when their tumors contain numerous cells expressing the inducible isoform of NO synthase (iNOS), and elevated levels of nitrotyrosine, a product derived from nitric oxide (NO). Although several lines of evidence suggest that NO promotes tumor growth and increases resistance to chemotherapy, it is unclear how it shapes these outcomes. Here we demonstrate that modulation of NO-mediated S-nitrosation of cellular proteins is strongly associated with the pattern of response to the anti-cancer agent cisplatin in human melanoma cells in vitro. Cells were shown to express iNOS constitutively, and to generate sustained nanomolar levels of NO intracellularly. Inhibition of NO synthesis or scavenging of NO enhanced cisplatin-induced apoptotic cell death. Additionally, pharmacologic agents disrupting S-nitrosation markedly increased cisplatin toxicity, whereas treatments favoring stabilization of S-nitrosothiols decreased its cytotoxic potency. Activity of the pro-apoptotic enzyme caspase-3 was higher in cells treated with a combination of cisplatin and chemicals that decreased NO/S-nitrisothiols, whereas lower activity resulted from cisplatin combined with stabilization of S-nitrisothiols. Constitutive protein S-nitrosation in cells was detected by analysis with biotin switch and reduction/chemiluminescence techniques. Moreover, intracellular NO concentration increased significantly in cells that survived cisplatin treatment, resulting in augmented S-nitrosation of caspase-3 and prolyl-hydroxylase-2 (PHD2), the enzyme responsible for targeting the pro-survival transcription factor HIF-1α for proteasomal degradation. Because activities of these enzymes are inhibited by S-nitrosation, our data thus indicate that modulation of intrinsic intracellular NO levels substantially affects cisplatin toxicity in melanoma cells. The underlying mechanisms may thus represent potential targets for adjuvant strategies to improve the efficacy of chemotherapy. Citation Format: Luiz C. Godoy, Chase C.T. Anderson, Rajdeep Chowdhury, Laura J. Trudel, Gerald N. Wogan. Melanoma fights cisplatin with NO: S-nitrosation as a mechanism supporting drug-resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 894. doi:10.1158/1538-7445.AM2013-894

Endogenously produced nitric oxide mitigates sensitivity of melanoma cells to cisplatin

Melanoma patients experience inferior survival after biochemotherapy when their tumors contain numerous cells expressing the inducible isoform of NO synthase (iNOS) and elevated levels of nitrotyrosine, a product derived from NO. Although several lines of evidence suggest that NO promotes tumor growth and increases resistance to chemotherapy, it is unclear how it shapes these outcomes. Here we demonstrate that modulation of NO-mediated S-nitrosation of cellular proteins is strongly associated with the pattern of response to the anticancer agent cisplatin in human melanoma cells in vitro. Cells were shown to express iNOS constitutively, and to generate sustained nanomolar levels of NO intracellularly. Inhibition of NO synthesis or scavenging of NO enhanced cisplatin-induced apoptotic cell death. Additionally, pharmacologic agents disrupting S-nitrosation markedly increased cisplatin toxicity, whereas treatments favoring stabilization of S-nitrosothiols (SNOs) decreased its cytotoxic potency. Activity of the proapoptotic enzyme caspase-3 was higher in cells treated with a combination of cisplatin and chemicals that decreased NO/SNOs, whereas lower activity resulted from cisplatin combined with stabilization of SNOs. Constitutive protein S-nitrosation in cells was detected by analysis with biotin switch and reduction/chemiluminescence techniques. Moreover, intracellular NO concentration increased significantly in cells that survived cisplatin treatment, resulting in augmented S-nitrosation of caspase-3 and prolyl-hydroxylase-2, the enzyme responsible for targeting the prosurvival transcription factor hypoxia-inducible factor-1α for proteasomal degradation. Because activities of these enzymes are inhibited by S-nitrosation, our data thus indicate that modulation of intrinsic intracellular NO levels substantially affects cisplatin toxicity in melanoma cells. The underlying mechanisms may thus represent potential targets for adjuvant strategies to improve the efficacy of chemotherapy.

Abstract 2958: The novel protein kinase STK17A is a direct p53 target gene that mediates response to genotoxic and nutritional stress in a cancer cell context-dependent manner

Abstract STK17A (DRAK1) is a largely uncharacterized serine/threonine kinase that belongs to the death-associated protein kinase family. Prior microarray studies demonstrated that STK17A is induced with cisplatin in testicular cancer-derived human embryonal carcinoma (EC) cells. We now demonstrate the STK17A in a novel p53 target gene that is induced by a variety of DNA damaging agents in a p53-dependent manner in a number of different cell contexts. A consensus and functional p53 response element was found upstream of the STK17A promoter and endogenous p53 was shown to bind to this site in a cisplatin-dependent manner. Knockdown of STK17A conferred resistance to cisplatin-induced growth suppression and apoptotic cell death in EC cells. This was associated with up-regulation of detoxifying and antioxidant genes and reduction in reactive oxygen species (ROS), whereas overexpression of STK17A increased ROS levels in EC cells. Interestingly, we found that STK17A is highly overexpressed in clinical glioblastoma (GBM) and cell lines compared to its expression in normal brain tissue and other cancers. High STK17A expression in GBM patients correlated with poor clinical outcome and decreased survival. STK17A knockdown in GBM cells decreased clonagenic cell growth, soft agar tumorigenicity, and survival in response to genotoxic and nutritional stress. Preliminary data will be presented implicating STK17A in the regulation of autophagy and the proximal autophagy component, ULK1. These findings indicate that STK17A may be added to a growing list of direct p53 target genes involved in autophagy that includes TIGAR, Sestrins, DAPK1 and DRAM and that by impacting autophagy STK17A may modulate response to genotoxic and nutritional stress in a cell context-depend manner. This study also suggests that STK17A is a potential novel prognostic maker and therapeutic target for GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2958. doi:1538-7445.AM2012-2958

Abstract 4899: Altered receptor glycosylation confers cisplatin resistance in ovarian and colon cancer cells

Abstract Chemotherapy resistance is a significant clinical obstacle in the treatment of cancer. Platinum-based drugs are a mainstay of treatment in many solid cancers; however, tumor resistance to these drugs hampers long term efficacy. Resistance is multi-factorial, and theories to explain resistance are incomplete. In this study we report a novel mechanism for resistance to cisplatin involving changes in receptor glycosylation. Previous work in our lab has demonstrated enhanced colon cancer cell survival in cells expressing ST6Gal-I, a Golgi glycosyltransferase that adds the negatively-charged sugar, alpha2-6-linked sialic acid, to selected membrane proteins. We showed that sialylation of the Fas death receptor by ST6Gal-I decreases apoptotic signaling in response to Fas agonists by blocking DISC formation and receptor internalization. Elevated ST6Gal-I expression has been reported in many cancers, including ovarian and colon, and its expression is negatively correlated with patient prognosis. Interestingly, cisplatin has been demonstrated to mediate its cytotoxic effects, in part, through Fas receptor upregulation and activation, although the mechanism is not currently understood. Combining these results and observations, we investigated whether ST6Gal-I-mediated sialylation of the Fas receptor inhibits apoptotic signaling in response to cisplatin treatment, contributing to intrinsic cisplatin resistance. We determined the extent of cisplatin-induced apoptotic cell death by blotting for cleaved caspase-3, a terminal caspase, and detected decreased levels of activated caspase in ST6Gal-I expressing ovarian and colon cell lines. This result was observed in both a low endogenous ST6Gal-I cell model with forced ST6Gal-I expression, as well as a high endogenous ST6Gal-I expression cell model with shRNA ST6Gal-I knockdown. We also show sustained viability in cells expressing ST6Gal-I after cisplatin treatment by measuring a peptide protease substrate which is cleaved in live cells. Conversely, we detected a decrease in viability in non-ST6Gal-I expressing counterpart cell lines indicating that cells lacking ST6Gal-I expression are more sensitive to cisplatin-mediated cell death. Finally, we observed that cisplatin-mediated cell death is attenuated by treating cells with blocking antibodies to Fas, consistent with other studies implicating this receptor in cisplatin efficacy. Our findings suggest a novel mechanism contributing to platinum drug resistance in cancer cell populations and identify ST6Gal-I as a promising target for therapeutic intervention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4899. doi:1538-7445.AM2012-4899

Cdc25A promotes cell survival by stimulating NF-κB activity through IκB-α phosphorylation and destabilization

Cell division cycle 25A (Cdc25A), a dual specificity protein phosphatase, exhibits anti-apoptotic activity, but the underlying molecular mechanisms are poorly characterized. Here we report that Cdc25A inhibits cisplatin-induced apoptotic cell death by stimulating nuclear factor-kappa B (NF-κB) activity. In HEK-293 cells, Cdc25A decreased protein level of inhibitor subunit kappa B alpha (Iκ-Bα) in association with increased serine 32-phosphorylation, followed by stimulation of transcriptional activity of NF-κB. Inhibition of NF-κB activity by chemical inhibitor or overexpression of Iκ-Bα in Cdc25A-elevated cancer cells resistant to cisplatin improved their sensitivity to cisplatin-induced apoptosis. Our data show for the first time that Cdc25A has an important physiological role in NF-κB activity regulation and it may be an important survival mechanism of cancer cells.

Protective effects of apocynin on cisplatin‐induced ototoxicity in an auditory cell line and in zebrafish

Cisplatin is a very effective anticancer drug and generates reactive oxygen species (ROS) such as superoxide anions that can deplete antioxidant protective molecules in the cochlea. These processes result in the death of cochlear hair cells by induction of apoptosis. Apocynin, which is used as a specific nicotinamide adenine dinucleotide phosphate oxidase inhibitor, has a preventive effect for intracellular ROS generation. In this study, the effect of apocynin was investigated in a cochlear organ of Corti-derived cell line, HEI-OC1 cells, and in transgenic zebrafish (Brn3C: EGFP). To investigate the protective effects of apocynin, HEI-OC1 cells were treated with various concentrations of apocynin and a 20 µm concentration of cisplatin, simultaneously. An in vivo study of transgenic zebrafish (Brn3C: EGFP) was used to investigate the protective effects of apocynin on cisplatin-induced hair cell death. In an in vitro study, apocynin appeared to protect against cisplatin-induced apoptotic features on Hoechst 33258 staining in the HEI-OC1 cells. Treatment of the HEI-OC1 cells with 100 µm of apocynin, significantly decreased caspase-3 activity. Treatment of the cells with a 100 µm concentration of apocynin and a 20 µm concentration of cisplatin significantly decreased the intracellular ROS production. In the in vivo study, apocynin significantly decreased the TUNEL reaction and prevented cisplatin-induced hair cell loss of the neuromasts in the transgenic zebrafish at low concentrations (125 and 250 µm). These findings suggest that apocynin has antioxidative effects and prevents cisplatin-induced apoptotic cell death in HEI-OC1 cells as well as in zebrafish.