Vy Dinh

N' 1 ,N' 3 -Dimethyl-N' 1 ,N' 3 -bis(phenylcarbonothioyl) Propanedihydrazide (Elesclomol) Selectively Kills Cisplatin Resistant Lung Cancer Cells through Reactive Oxygen Species (ROS)

Cisplatin is an important chemotherapeutic agent in lung cancer treatment. The mechanism of drug resistance to cisplatin is complex and historically has been difficult to overcome. We report here that cisplatin resistant lung cancer cell lines possess high basal levels of reactive oxygen species (ROS) when compared to normal cells and their parental cell counterparts. These resistant cells also have low thioredoxin (TRX) levels which may be one of the contributory factors to high ROS. N′1,N′3-dimethyl-N′1,N′3-bis(phenylcarbonothioyl) propanedihydrazide (elesclomol), an agent known to increase ROS is selectively toxic to cisplatin-resistant cells, while sparing normal cells and the parental counterpart. The cytotoxic effect of elesclomol in resistant cells is accompanied by further decreases in TRX and glutathione (GSH) antioxidant systems, while opposite results were found in parental cells. The ID50 of elesclomol in cisplatin-resistant cells ranged from 5–10 nM, which is well within clinically achievable ranges. N-Acetylcysteine (NAC), which is known to neutralize ROS, can abolish the cytotoxic effect of elesclomol, suggesting that the cytotoxic effect results from increased ROS. Overall, our data suggest that elesclomol selectively kills cisplatin-resistant tumor cells through increased ROS. This agent may hold potential to overcome cisplatin resistance and should be further explored to treat patients who have failed cisplatin therapy.

Abstract 61: Enhancing arginine deprivation therapy in melanoma by combining with cisplatin

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC We have previously shown that arginine deprivation using pegylated arginine deiminase (ADI-PEG20) has activity in melanoma patients whose tumors do not express argininosuccinate synthetase (ASS). However, not all ASS-negative patients respond to treatment, and relapse does occur. In our laboratory, we have found that there are two major mechanisms of resistance to ADI-PEG20: 1) induction of ASS expression upon arginine deprivation and 2) the ability of ASS-negative cells to undergo autophagy and hence evade apoptosis. These cells often have high level of anti-apoptotic proteins such as Bcl-2. To overcome resistance to ADI-PEG20, one can add an agent to either accelerate cell death before cells are rescued by induction of ASS expression or channel cells directly to apoptosis instead of autophagy. Cisplatin is known to induce apoptosis via the mitochondria pathway while ADI-PEG20 can also induce apoptosis through nutritional stress via the same pathway. Therefore combination of these two agents should intensify the apoptotic signal. We have investigated this possibility using a panel of six melanoma cell lines (A375, A2058 and SK-Mel-2 from ATCC; Mel-1220, Mel-F, and Mel-114 established in our laboratory). We have found that the combination of cisplatin (0.1 ug/ml) with ADI-PEG20 (0.05 ug/ml) can effectively induce cell death in all melanoma cell lines tested. The growth inhibition increased from 30-40% with ADI-PEG20 alone to 60-70% with combination in the ATCC cell lines (p < 0.001), and from 35% to 50% in our own cell lines (p =< 0.05). When treated with cisplatin alone at the same dosage, the growth inhibition was only 10-20% for all these cell lines. This combination also increased the percentage of cells undergoing apoptosis. When compared with cisplatin alone treatment, the percentage of cells with caspase activation increased from 5% (0.5 ug/ml of cisplatin) to 29% (combination with ADI-PEG20 at 0.05 ug/ml), or from 20% (1 ug/ml of cisplatin) to 75% (combination with ADI-PEG20 at 0.05 ug/ml) in A375. The increase of cell death was less (12-15% to 25-30%) in Mel-1220 and Mel-F which have high levels of Bcl-2 protein. This combination treatment is not schedule-dependent. In our study of the combination treatment, we found that the combination led to more DNA damage than cisplatin alone in Mel-1220 as revealed by γ-H2AX immunofluorescence. This combination also increased the expression of pro-apoptotic protein Noxa, and resulted in decrease of anti-apoptotic protein Survivin in Mel-1220 and A375. No apparent changes were detected for Bcl-2, Mcl-1, and XIAP. Interestingly, tBid formation was also observed in the combination treatment in A375 and SK-Mel-2 which also indicates the enhancement of apoptotic signal when cisplatin and ADI-PEG20 were combined. From these data, the combination of cisplatin and ADI-PEG20 should be a more effective treatment for melanomas which lack ASS expression. Supported by NIH grant 1RO1CA109578. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 61.

Abstract 1038: Exploiting ROS and metabolic differences to selectively killing cisplatin resistant lung cancer

Cisplatin resistance remains a major problem in the treatment of lung cancer. We have discovered that cisplatin resistant (CR) lung cancer cells, regardless of the KRAS mutation status, share one common parameter which is increased reactive oxygen species (ROS) when compared to their parental cells counterpart. Importantly, we found that CR cells were no longer addicted to the glycolytic pathway, but rather relied on oxidative metabolism (OXMET) for energy and biosynthesis. Thus, alteration in mitochondria which resulted in metabolic changes and higher basal levels of ROS may be one of the major factors contributing to cisplatin resistance. We have investigated mitochondrial morphology in parental vs. CR cells using transmission electron microscopy (TEM). Our data clearly showed that CR cells possessed significantly higher number of mitochondria per total cell area (n = 4; p = 0.0006). CR cells also consumed 2-4 fold more oxygen when compared to their parental cells. To further confirm that oxidative phosphorylation was utilized more in CR cells, we assayed mitochondrial membrane potential (MMP) using TMRE staining. MMP was significantly higher in CR cells suggesting that CR cells possess highly active mitochondria. Key glycolytic enzymes hexokinase II (HKII) and lactate dehydrogenase A (LDHA), as well as lactate production were decreased in all CR cell lines which corresponded to the resistance to glycolytic inhibitor, 2DG (n = 8; p Citation Format: Medhi Wangpaichitr, Chunjing Wu, Ying Ying Li, Sumedh Shah, Shu-Mei Chen, Vy Dinh, Macus T. Kuo, Lynn G. Feun, Niramol Savaraj. Exploiting ROS and metabolic differences to selectively killing cisplatin resistant lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1038.

Abstract 43: The role of discoidin domain receptor 2 (DDR2) and its relationship with procollagen alpha 1 type 1(Col1A1) in malignant glioma

We have previously shown that Col1A1 expression is found more frequently in low to intermediate grade glioma than high grade glioma(Cancer invest. 23:577,2005). On the other hand, its receptor DDR1 is found in high grade glioma and often signifies poor prognosis. We hypothesize that DDR2 which also binds to Col1A1 may play a role in mediating this complex relationship. We have investigated this relationship using 3 cell lines (2 cell lines, Glioma 1 and U-118, both express both Col1A1 and DDR1&2, A-172 which does not express Col1A1 but does express both DDR1&2). All cell lines were maintained in EMEM with 10% FCS. Treatment with Befeldin A (BFA), which blocks Col1a1 to the cell surface, results in ER stress with increase in GRP78 and growth inhibition in all three cell lines. U-118 was the most sensitive (ID50 of 9 ng/ml for U- 118, Glioma 1 (13ng/ml), and A-172 (24ng/ml)). Silencing Col1A1 resulted in decreased DDR1 & 2 in Glioma 1 and U-118 and decreased sensitivity to BFA by 20% for Glioma 1 and 10% in U-118. Silencing Col1 A1 resulted in G2M arrest and decrease in ERK1/2, JNK, and c-Jun, but increase in PDGF, CREB and P70 by protein array analysis and confirmed by Western blot. We further investigated the biological effect of silencing either DDR1 (primary DDR1a which is the major isoform found in Glioma 1 and DDR2). We found that silencing DDR 1 had no effect on DDR2 expression and vice versa. Interestingly, in contrast to silencing Col1A1, silencing either DDR1 or 2 had no effect on Col1A1 expression in Glioma 1 and U-118 and also no effect on cell cycle or cellular proliferation. In addition, silencing DDR1 or 2 led to decreased sensitivity to BFA for both Glioma and U-118 with increase in ID50 by 35-50% respectively. Furthermore, we have also found an increase in p-AKT, p-38, JNK and p-Jun after 48-hr post silencing of DDR2 while silencing DDR1 had minor effect. However, these proteins decreased after BFA treatment. In contrast, silencing DDR1 or DDR2 led to increased sensitivity to BFA in A-172 cells with reduction of ID50 by 20%. In addition, a decrease in p-AKT, p-38 and ERK were also seen. Silencing DDR1 or DDR2 also showed significant reduction in invasion in A-172 cell line, while minimal changes were seen in Glioma 1 and U-118. Since DDR1 has multiple subfamilies, while DDR1a is the most common one in brain tumor, it is possible that silencing one subfamily may upregulate the other subfamily. However, DDR2 has only one family and may explain the more profound effect in silencing DDR2. Taken together, our data suggest that the function of DDR2 is different among cell lines, and may be dictated by the presence of Col1A1 which is present in certain malignant glioma. We are currently investigating the possible mechanism(s) involved. Future targeted therapy in malignant glioma will need to take into consideration the different functions of DDR2. (Supported by Wanfang Hospital-Taipei medical university fellowship award and VA research grant) Citation Format: Shumei Chen, Chunjing Wu, Ying Ying Li, Medhi Wangpaichitr, Lynn G. Feun, Ronald Benveniste, Vy Dinh, Niramol Savaraj. The role of discoidin domain receptor 2 (DDR2) and its relationship with procollagen alpha 1 type 1(Col1A1) in malignant glioma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 43. doi:10.1158/1538-7445.AM2015-43

Abstract 1428: Targeting metabolic alteration in cisplatin-resistant lung cancer

We have discovered that all cisplatin resistant (CR) lung cancer cells are less reliant on glycolysis while increasing oxidative metabolism and mitochondrial numbers. Here, we showed that glutamine withdrawal has a detrimental effect on CR cell lines, especially in non small cell lung cancer (NSCLC) CR cells. We assayed for glutaminase, a key enzyme in glutaminolysis pathway and found a significant decrease in protein expression and activity in all our CR cells (n=8, P 0.05). Since not all CR cells exhibited similar sensitivity to glutamine withdrawal, we further explored other key amino acids which may be used in TCA cycle. We have found that NSCLC-CR lost argininosuccinate synthetase (ASS) expression while SCLC-CR showed an increase. ASS is a key enzyme in urea cycle to regenerate arginine. Thus, NSCLC-CR will depend on exogenous arginine for their survival. Moreover, arginine in the urea cycle can be used to generate glutamate via proline pathway using P5CDH (pyrroline-5-carboxylate dehydrogenase) which may explain why NSCLC-CR are auxotrophic for both arginine and glutamine. To investigate this possibility, we have studied P5CDH expression in all CR cells and their parental cells in complete and glutamine-free media. We have found that NSCLC-CR has significantly less to non-detectable levels of P5CDH and cannot be upregulated upon glutamine deprivation, while its parental cells have higher P5CDH expression which is further upregulated on glutamine deprivation. In contrast, SCLC-CR has significantly higher P5CDH expression compared to its parental cells and both can be upregulated upon glutamine deprivation. To further confirm this finding, we have overexpressed ASS using the pCMV6 vector containing full length ASS cDNA in NSCLC cells (SCASS). These transfected cells were able to withstand arginine free media compared to the NSCLC-CR transfected with empty vector (10% of NSCLC-CR cells were viable after 72hrs in arginine free media compared to 90% of SCASS were still viable). SCASS also took up less glutamine and was able to withstand glutamine free media when compared to NSCLC-CR. Furthermore, SCASS expressed higher levels of P5CDH and was further upregulated upon glutamine withdrawal. Our data demonstrate a complex metabolic network between arginine, proline, and glutamine. The derangement of the key enzymes which are necessary for generation of these amino acids can be exploited for future clinical application to selectively kill cisplatin resistant cells and improve treatment outcome in lung cancer patients. Supported by Department of Veterans Affairs, CDA2 award. Citation Format: Medhi Wangpaichitr, Chunjing Wu, Ying Ying Li, Shumei Chen, Min You, Vy Dinh, Lynn G. Feun, Macus T. Kuo, Niramol Savaraj. Targeting metabolic alteration in cisplatin-resistant lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1428. doi:10.1158/1538-7445.AM2014-1428

Abstract 4052: Targeting procollagen secretory pathway for the treatment of primary brain tumor through ER stress.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC We have previously shown that primary brain tumor from patients and cell lines possess type I alpha 1 procollagen. Procollagen type I, III, and IV have also been reported to be expressed in certain glioma subtypes. Although the function of these procollagens is not known, they are most likely involved in invasion and signaling. We have further investigated whether manipulation of the secretion of these procollagens can be used to kill glioma cells. We have studied the expression of procollagen type I alpha 1 and2 (Pro-col1A1 & A2), procollagen type III alpha 1 (Pro-col3A1) and procollagen type IV alpha 1&2 (Proc-col4A1 & A2) in 5 cell lines (Glioma-1, U-373, U-118, U-87, and A-172) using real time RT-PCR (qPCR) and western blot when antibody was available. Our data showed that A-172 expressed all 5 types of procollagen, while U-87, U-118 and Glioma-1 expressed primary Pro-colA1 and A2, and U-373 expressed only pro-col1A2 and pro-col4A1 and A2. Hsp47, which is a major chaperone in the folding of procollagen type I, is not expressed in U-373. This cell line also possessed very high levels of GRP-78 which is indicative of increased ER stress. We also investigated the presence of collagen receptor Endo 180 and DDR1 (discodin domain receptor 1) in these cell lines. Endo 180 was present in all 5 cell lines, but to a much lesser extent in U-373. The DDR1 expression by qPCR was the highest in A-172 followed by U-118 (>5 arbitrary units) while U-373, U-87 and Glioma-1 have less than 3 arbitrary units. It is known that procollagen needs to be folded at ER and then secreted out of the cells, where it will be processed to form collagen. Collagen can then bind DDR1, a receptor tyrosine kinase which plays a role in attachment, migration, proliferation and survival. In this regard, we have detected pro-col1A1 and A2 in the media which verified that pro-col1A1 and A2 are secreted outside the cells. We hypothesize that inability to secrete procollagen will result in both ER stress and inability for signaling. To accomplish this, we have studied the cytotoxic effect of brefeldin (BFA) which is known to block the transport of secretory protein from ER/Golgi network to the cell surface in these five cell lines. All five cell lines are very sensitive to brefeldin with IC50 of 3.0 ng/ml for U-118, A-172 and U-87. In U-373 and Glioma-1 the IC50 was 5.0 ng/ml. Interestingly, cancer stem cells isolated from U-87 did not possess pro-co1A1, but DDR1 mRNA was present and was 3 times higher than in the non stem cells. They also have different metabolic profiling than their parental cells, such as they do not express argininosuccinate synthetase, a key enzyme to synthesize arginine which made them sensitive to arginine deprivation treatment. Whether these differences exist in vivo is not known. Overall, our data indicate that the expression of procollagen and its secretory pathway can be used as a new paradigm to treat primary brain tumor through ER stress-mediated cell death. Citation Format: Min You, Shu-Mei Chen, Chunjing Wu, Ying-Ying Li, Lynn Feun, Medhi Wangpaithitr, Vy Dinh, Niramol Savaraj. Targeting procollagen secretory pathway for the treatment of primary brain tumor through ER stress. [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 4052. doi:10.1158/1538-7445.AM2013-4052

Targeting Argininosuccinate Synthetase in Cancer Therapy

There has been renewed interest in amino acid deprivation to treat various human cancers. Certain tumors do not express argininosuccinate synthetase (ASS) and therefore are unable to synthesize arginine from citrulline. These tumors are auxotrophic for arginine and may be inhibited by arginine deprivation. Arginine deprivation can affect growth and apoptotic signaling. In addition, mTOR signaling and RAF/MEK/ERK1/2 signaling are also affected by arginine deprivation. Upon arginine deprivation, tumor cells undergo autophagy as a survival mechanism, but prolonged autophagy can lead to apoptotic cell death. Arginine derivation using pegylated arginine deiminase (ADI-PEG20) has been shown to have activity in malignant melanoma and hepatocellular carcinoma with minimal side effects. Why ASS is silent in certain tumor types is not known. Aberrant DNA methylation which leads to epigenetic silencing has been reported. Reexpression of ASS may lead to resistance to arginine deprivation treatment. Thus, understanding how the ASS gene is regulated is very important for this modality of cancer treatment.

Abstract 1692: Downregulation of thioredoxin-1 confers resistance to cisplatin and sensitivity to the ROS generating agent elesclomol

We have previously discovered a unique and important finding that all of our cisplatin resistant (CR) small cell and non small cell lung cancer cells (5 pairs of cell lines, parental vs. CR cells, and one primary line tested) possess high levels of ROS (Reactive Oxygen Species) compared to their parental cancer cell counterparts and normal cells. Importantly, these CR cells are sensitive to elesclomol (Synta Pharmaceuticals), a new compound which kills cells by generating ROS (Cancers, 23-28; 2009). The question remains why these CR cells possess intrinsically high ROS levels. We have found that the common biochemical feature in CR cells is lower expression of the antioxidant protein thioredoxin-1 (TRX1). CR cells have an average of 6-8 fold lower TRX1 as compared to their parental counterparts and normal cells. Other antioxidant systems either showed no changes or elevated such as GSH but varies among cell lines. To further verify that TRX1 is an important contributory factor to the high ROS levels been in CR cells, we knocked down TRX1 in parental cells (SCLC1) using siRNA. These SCLC1/TRX1(−) transfectants generated significantly higher ROS (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1692. doi:10.1158/1538-7445.AM2011-1692

Abstract 4525: Targeting ROS to kill cisplatin-resistant cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Cisplatin (CP) resistance remains a major problem in the treatment of both small cell and non small cell lung cancer. We have previously shown that inhibiting mTOR can restore CP sensitivity; however, not all CP resistant cell lines are sensitive to mTOR inhibitor. Our findings have led us to search for an alternative target in these CP resistant cells. We have discovered that CP resistant cells share one common biochemical parameter which is increased reactive oxygen species (ROS) when compared to normal cells and their parental cells counterpart. Thus, further increased ROS in these CP resistant cells can push them beyond their tolerance limit which ultimately leads to cell death. We have found that increased expression of NADPH oxidase 4 (NOX4) and lower levels of thioredoxin (Trx) correlate with the high levels of ROS seen in CP resistant cell lines. Using elesclomol (N′1, N′3-dimethyl-N′1, N′3- bis [phenylcarbonothioyl] propanedihydrazide (formerly STA-4783, provided by Synta Pharmaceuticals)), an agent which is known to increase ROS, we demonstrated that elesclomol can increase H2O2 levels in CP resistant lung cancer cell lines (CP-R). Moreover, elesclomol is also significantly more cytotoxic toward CP-R with the ID50 dosage of 4-10 fold less than their parental cells (PC) counterpart and normal cells, BJ-1 (see table below). The percentage of apoptosis is also significantly higher in CP-R (65%) vs. PC (3%). The cytotoxic effect of elesclomol in CP-R is accompanied by further decrease in Trx and glutathione (GSH) antioxidant systems, while opposite results were found in PC and normal cells. This cytotoxic effect can be reversed by an antioxidant, N-acetylcysteine (NAC, 0.1mM). Furthermore, we have also found that elesclomol also increased intracellular CP and hence enhanced CP sensitivity in CP-R cells. We conclude that elesclomol is highly effective in CP resistant cells which express high basal levels of ROS and should be considered for treatment of CP resistant tumor. View this table: ID50 Dosage of Elesclomol in Parental (PC) vs. Cisplatin Resistant (CP-R) Lung Cancer Cell Lines Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4525.