Subbulakshmi Virudachalam

Targeting BCL-2 overexpression in various human malignancies through NF-κB inhibition by the proteasome inhibitor bortezomib

BackgroundBCL-2 overexpression occurs in many cancer types and is associated with chemoresistance and radioresistance. The mechanisms responsible for its aberrant expression are thought to be transcriptionally mediated but remain unclear. We examined the cell type-specific mechanism of BCL-2 gene transcription in various solid organ malignancies.MethodsRegulation of BCL-2 gene transcription was examined in seven different human cancer cell lines including two pancreatic (MIA-PaCa-2, PANC-1), two prostate (LNCaP, PC-3), two lung (Calu-1, A549) and one breast (MCF-7) cancer cell line. Cells were treated with inhibitors of phosphatidylinositol-3 kinase (PI3K), MEK/ERK, and p38MAPK. The effect of mutation of a NF-κB site in the BCL-2 promoter was determined, as was the effect of inhibition of NF-κB function using a 26S proteasome inhibitor (bortezomib) on both BCL-2 transcription and induction of apoptosis.ResultsBCL-2 expression varied both between and within tumor types; four of seven cell lines demonstrated high BCL-2 levels (MIA-PaCa-2, PC-3, Calu-1 and MCF-7). No signaling pathway was uniformly responsible for overexpression of BCL-2; however, mutation of the NF-κB site decreased BCL-2 promoter activity in all cell lines. Inhibition of NF-κB activity decreased BCL-2 protein levels independently of the signaling pathway involved in transcriptional activation of the BCL-2 gene.ConclusionsDiverse signaling pathways variably regulate BCL-2 gene expression in a cell type-specific fashion. Therapy to decrease BCL-2 levels in various human cancers would be more broadly applicable if targeted to transcriptional activation rather than signal transduction cascades. Finally, the apoptotic efficacy of proteasome inhibition with bortezomib paralleled the ability to inhibit NF-κB activity and decrease BCL-2 levels.

AKT inhibition is associated with chemosensitisation in the pancreatic cancer cell line MIA-PaCa-2

Activation of the serine/threonine kinase AKT is common in pancreatic cancer; inhibition of which sensitises cells to the apoptotic effect of chemotherapy. Of the various downstream targets of AKT, we examined activation of the NF-κB transcription factor and subsequent transcriptional regulation of BCL-2 gene family in pancreatic cancer cells. Inhibition of either phosphatidylinositol-3 kinase or AKT led to a decreased protein level of the antiapoptotic gene BCL-2 and an increased protein level of the proapoptotic gene BAX. Furthermore, inhibition of AKT decreased the function of NF-κB, which is capable of transcriptional regulation of the BCL-2 gene. Inhibiting this pathway had little effect on the basal level of apoptosis in pancreatic cancer cells, but increased the apoptotic effect of chemotherapy. The antiapoptotic effect of AKT activation in pancreatic cancer cells may involve transcriptional induction of a profile of BCL-2 proteins that confer resistance to apoptosis; alteration of this balance allows sensitisation to the apoptotic effect of chemotherapy.

Effects of the proteasome inhibitor bortezomib alone and in combination with chemotherapy in the A549 non-small-cell lung cancer cell line.

BackgroundNon-small-cell lung cancer (NSCLC) has a poor prognosis. Despite advances in therapy, survival has improved only slightly. The 26S proteasome regulates multiple cellular processes through degradation of ubiquitin-tagged proteins. The proteasome inhibitor, bortezomib (Velcade, formerly PS-341), has been shown to be an active anticancer agent both in vitro and in vivo in multiple tumor types.PurposeTo determine the molecular and cellular effects of the proteasome inhibitor in NSCLC as well as to evaluate the effectiveness of sequential treatment with bortezomib and gemcitabine/carboplatin (G/C) chemotherapy both in vitro and in vivo.MethodsAll experiments were performed in the A549 NSCLC cell line. MTT assays were used to evaluate cytotoxicity. Western blotting evaluated protein levels. Measures of apoptosis included FACS analysis, DAPI staining and caspase-3 cleavage. Long-term cell viability was determined using an anchorage-dependent clonogenic assay. Sequential studies were performed in vitro and in vivo.ResultsBortezomib increased p21waf1/cip1, induced G2/M arrest, and triggered a small amount of apoptosis. The apoptotic effect of G/C chemotherapy was eliminated when bortezomib was administered prior to the chemotherapy; however, it was accentuated when the bortezomib was given simultaneously or after the chemotherapy.ConclusionsBortezomib improves efficacy in combination with gemcitabine and carboplatin in NSCLC, but sequential effects are important and must be considered when developing therapeutic regimens.

Pancreatic cancer cell lines deficient in argininosuccinate synthetase are sensitive to arginine deprivation by arginine deiminase

Eukaryotic cells can synthesize the non‐essential amino acid arginine from aspartate and citrulline using the enzyme argininosuccinate synthetase (ASS). It has been observed that ASS is underexpressed in various types of cancers ASS, for which arginine become auxotrophic. Arginine deiminase (ADI) is a prokaryotic enzyme that metabolizes arginine to citrulline and has been found to inhibit melanoma and hepatoma cancer cells deficient of ASS. We tested the hypothesis that pancreatic cancers have low ASS expression and therefore arginine deprivation by ADI will inhibit cell growth. ASS expression was examined in 47 malignant and 20 non‐neoplastic pancreatic tissues as well as a panel of human pancreatic cancer cell lines. Arginine deprivation was achieved by treatment with a recombinant form of ADI formulated with polyethylene glycol (PEG‐ADI). Effects on caspase activation, cell growth and cell death were examined. Furthermore, the effect of PEG‐ADI on the in vivo growth of pancreatic xenografts was examined. Eighty‐seven percent of the tumors lacked ASS expression; 5 of 7 cell lines similarly lacked ASS expression. PEG‐ADI specifically inhibited growth of those cell lines lacking ASS. PEG‐ADI treatment induced caspase activation and induction of apoptosis. PEG‐ADI was well tolerated in mice despite complete elimination of plasma arginine; tumor growth was inhibited by ∼50%. Reduced expression of ASS occurs in pancreatic cancer and predicts sensitivity to arginine deprivation achieved by PEG‐ADI treatment. Therefore, these findings suggest that arginine deprivation by ADI could provide a beneficial strategy for the treatment of pancreatic cancer, a malignancy in which new therapy is desperately needed.

ERK/BCL-2 pathway in the resistance of pancreatic cancer to anoikis.

BACKGROUND Anoikis is a special type of programmed cell death after loss of cell-cell and cell-extracellular matrix interactions. Resistance to anoikis is likely involved in the process of metastasis, specifically during the tumor cell migration through lymph or vascular channels. We have previously shown that BCL-2 confers resistance to other forms of programmed cell death (i.e., apoptosis); furthermore, the extracellular signaling-regulated kinase (ERK) signaling pathway regulates BCL-2 expression. We therefore tested the hypothesis that pancreatic cancer cell lines are resistant to anoikis and this resistance is due to activation of ERK1/2 and subsequent overexpression of BCL-2. MATERIALS AND METHODS Pancreatic cancer cell lines (MIA-PaCa-2 and BxPC-3) were examined for cell death following loss of adherence to extracellular matrix. Subclones of the MIA-PaCa-2 cell line (either selected in vivo for increased metastatic potential [MIA-LM2] or overexpressing BCL-2 [MIA-BCL2]) were also examined for induction of anoikis following loss of extracellular matrix adherence. Finally, the effect of the ERK inhibitor (PD98059) on BCL-2 expression and induction of anoikis was examined. RESULTS Under conditions of loss of cell-extracellular matrix interaction, pancreatic cancer cells undergo varying amounts of anoikis. Basal levels of activated ERK and BCL-2 paralleled the sensitivity to induction of anoikis. The highly metastatic cell line, MIA-LM2, was more resistant to anoikis than the parental cell line. Inhibition of ERK down-regulated BCL-2 and was associated with restoration of sensitivity to anoikis. CONCLUSIONS Activation of a signaling pathway from ERK to overexpression of BCL-2 may confer resistance to anoikis, a critical step in the development of metastasis. Targeting the ERK/BCL-2 pathway may lead to sensitization of pancreatic cancer to anoikis, thereby decreasing the ability of these cells to metastasize.

BCL-2 functions as an activator of the AKT signaling pathway in pancreatic cancer

BCL‐2 is the prototypic anti‐apoptotic protein involved in the regulation of apoptosis. Overexpression of BCL‐2 is common in pancreatic cancer and confers resistance to the apoptotic effect of chemo‐ and radiotherapy. Although these cellular effects of BCL‐2 are traditionally related to pathways involving the mitochondrial membrane, we sought to investigate whether BCL‐2 is involved in other signaling pathways regulating cell survival and focused on AKT. We examined the effect of overexpression of BCL‐2 in the MIA‐PaCa‐2 human pancreatic cancer cell line on the function and subcellular location of AKT. We observed that the stable subclones of MIA‐PaCa‐2 overexpressing BCL‐2 demonstrated increased activity of AKT as well as IKK (a downstream target of AKT), increasing the transcriptional activity of NF‐κB. Using immunoprecipitation techniques, we observed co‐immunoprecipitation of AKT and BCL‐2. Immunocytochemistry demonstrated co‐localization of BCL‐2 and AKT, which was abrogated by treatment with HA14‐1, a small molecule inhibitor of BH‐3‐mediated protein interaction by BCL‐2. Furthermore, treatment with HA14‐1 decreased phosphorylation of AKT and increased sensitivity to the apoptotic effect of the chemotherapeutic agent, paclitaxel. These results demonstrate an additional mechanism of regulation of cell survival mediated by BCL‐2, namely through AKT activation, in the MIA‐PaCa‐2 pancreatic cancer cell line. Therefore, directed inhibition of BCL‐2 may alter diverse pathways controlling cell survival and overcome the apoptotic resistance that is the hallmark of pancreatic cancer. J. Cell. Biochem. 102: 1171–1179, 2007.

Pegylated arginine deiminase synergistically increases the cytotoxicity of gemcitabine in human pancreatic cancer

BackgroundPancreatic ductal adenocarcinoma has proven to be one of the most chemo-resistant among all solid organ malignancies. Several mechanisms of resistance have been described, though few reports of strategies to overcome this chemo-resistance have been successful in restoring sensitivity to the primary chemotherapy (gemcitabine) and enter the clinical treatment arena.MethodsWe examined the ability of cellular arginine depletion through treatment with PEG-ADI to alter in vitro and in vivo cytotoxicity of gemcitabine. The effect on levels of key regulators of gemcitabine efficacy (e.g. RRM2, hENT1, and dCK) were examined.ResultsCombination of PEG-ADI and gemcitabine substantially increases growth arrest, leading to increased tumor response in vivo. PEG-ADI is a strong inhibitor of the gemcitabine-induced overexpression of ribonucleotide reductase subunit M2 (RRM2) levels both in vivo and in vitro, which is associated with gemcitabine resistance. This mechanism is through the abrogation of the gemcitabine-mediated inhibitory effect on E2F-1 function, a transcriptional repressor of RRM2.ConclusionThe ability to alter gemcitabine resistance in a targeted manner by inducing metabolic stress holds great promise in the treatment of advanced pancreatic cancer.

Targeting Bcl-2-Mediated Cell Death as a Novel Therapy in Pancreatic Cancer

BACKGROUND Bcl-2 is an essential regulator of programmed cell death (PCD). Overexpression of Bcl-2 is common in pancreatic cancer; the high levels have been shown to correlate with resistance to PCD. This resistance is mediated by binding of Bcl-2 via its BH-3 domain to diverse proteins, including the Bax/Bak family members, various protein kinases, and beclin 1, which are involved in regulation of autophagy (type II PCD). Small molecule inhibitors of BH-3-mediated binding of Bcl-2 have been recently developed, although no investigation has been conducted in pancreatic cancer, a malignancy characterized by extreme resistance to PCD. METHODS The effect of the Bcl-2 binding inhibitor A-779024 on PCD was assessed by fluorescence activated cell sorting; the effect on Bcl-2 and other PCD-related proteins was analyzed by immunoblotting. Induction of autophagy was determined by fluorescence microscopy using a stably transfected GFP-LC3 construct to visualize autophagosome formation. Co-localization of Bcl-2 with binding partners regulating PCD was examined by immunoprecipitation and confocal immunofluorescent microscopy. RESULTS A-779024 induced PCD in a dose- and time-dependent fashion. No change was seen in the protein levels of Bcl-2, Bax, Bcl-XL, or Mcl-1. Contrary to prediction, A-779024 was ineffective at inducing autophagy in these cells. Co-localization studies demonstrated that Bcl-2 was not bound to beclin 1 and, therefore, treatment with A-779024 could not induce release of beclin 1 and initiation of autophagy. CONCLUSIONS Disruption of Bcl-2 activity using the small molecule inhibitor A-779024 induces apoptotic but not autophagic PCD. This approach may be a novel therapy, either alone or in combination with other treatments such as chemotherapy or autophagy modulating agents in pancreatic cancer.

Abstract 3782: Chloroquine induces defective autophagic flux and enhances pancreatic cancer cell death induced by pegylated-arginine deiminase

We have previously shown that a majority of human pancreatic adenocarcinomas are deficient in argininosuccinate synthetase (ASS), a key enzyme in arginine synthesis and therefore sensitive to arginine deprivation by arginine deiminase (ADI). ASS deficient cell lines undergo cell death when deprived of arginine, but also undergo autophagy. The role of autophagy and its contribution to cell death is controversial. We hypothesized that autophagy is protective in the setting of arginine deprivation and that inhibition of autophagy by hydroxychloroquine (ChQ) would increase cell death. Methods: The human pancreatic cancer cell line MIA-PaCa2 was treated in vitro and in vivo with pegylated arginine deiminase (PEG-ADI) alone or in the presence of ChQ. Cell death was measured by propidium iodide-FACS and apoptosis was evaluated by Annexin V-PI flow cytometry. Caspase 3 cleavage was evaluated by western blotting and ELISA as was expression of LC3. In vivo experiments were conducted by creation of subcutaneous xenografts in athymic mice followed by treatment with intraperitoneal injections of PBS, PEG-ADI, ChQ and combination of PEG-ADI and ChQ. At sacrifice, tumors were removed for analysis. Tumor lysates were analyzed by western blot for caspase 3 and p62. Immunohistochemistry for activated caspase 3 and DNA fragmentation (TUNEL) were also performed. Results: In vitro treatment of MIA-PaCa2 with PEG-ADI induced caspase dependent cell death as well as autophagy at 72 hours. ChQ at low doses inhibited autophagy induced by PEG-ADI, but increased cell death. In vivo, addition of ChQ to a sub-therapeutic dose of PEG-ADI increased tumor suppression decreased autophagy and increased caspase 3 activation compared to either treatment alone. Conclusion: Arginine deprivation induces autophagy and cell death in cell lines deficient in ASS. Autophagy appears to play a protective role, and its inactivation by hydroxychloroquine leads to enhanced tumor suppression. 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 3782. doi:10.1158/1538-7445.AM2011-3782