Apoptosis In NSCLC Cells Research Articles

Abstract 974: Elucidating the synergistic action of histone deacetylase inhibitor and cisplatin in lung squamous cell carcinoma.

Abstract Non-small cell lung cancer (NSCLC) constitutes 85-90% of all lung cancer cases with squamous cell carcinoma (SCC) being one of the major subtypes of NSCLC (35%). Although the risk of developing lung SCC remains steadfast in recent years, it prevails as a major public health concern worldwide due to its low 5-year overall survival rate. Platinum-based chemotherapies are currently the standard of care NSCLC, however, resistance to platinum compounds pose to be a major obstacle in achieving long term therapeutic effects. Currently, the understanding on resistance in lung SCC is severely limited. While many targeted therapies have been recently approved for adenocarcinoma of the lung, only a limited few have been applied on lung SCC due to a paucity of identifiable driver mutations. Hence, elucidation of specific pathways responsible for platinum resistance may reveal novel chemotherapeutic targets aimed at circumventing resistance to platinum in lung SCC. From a panel of lung SCC cell lines, we categorized them into cisplatin-resistant and -sensitive groups as study models based on cell viability assays. Gene expression profiles in these models after cisplatin treatment revealed that genes involved in MAPK signalling pathway and phosphatidylinositol signalling system were down-regulated in the cisplatin-sensitive group. Further investigation utilizing phosphoprotein antibody arrays revealed changes to levels of phosphorylated signalling proteins, with phosphorylated Akt, Erk1/2, p38, GSK3 and RSK levels abrogated upon cisplatin treatment in a sensitive cell line. In contrast, elevated levels of phosphorylated Erk1/2 were observed in a resistant cell line, suggesting the involvement of Erk in cisplatin resistance. Inhibitors of histone deacetylase (HDAC) have been shown to induce differentiation, cell growth arrest, and apoptosis in NSCLC cells. Recent reports have demonstrated that HDAC inhibitors could re-sensitize tumours to chemotherapy, but the mechanism(s) for these observations remains unknown. Here, we observed that cisplatin-resistant SCC lines were more responsive towards PXD101, a pan-HDAC inhibitor. Interestingly, we observed that PXD101 selectively dephosphorylated Erk1/2 and GSK3α/β, but had no activity on phosphorylated Akt. Moreover, several SCC lines grown under anchorage-independent growth condition demonstrated that treatment with HDAC inhibitor could suppress the malignant growth of transformed cells in combination with cisplatin. Taken together, these results illustrate the synergistic effects of HDAC inhibitor in cisplatin-resistant lung SCC tumours. Our preliminary findings also convey that Erk1/2 could be a potential target for the treatment of lung SCC tumours with intrinsic resistance to cisplatin. Citation Format: Li Ren Kong, Kian Ngiap Chua, Lingzhi Wang, Wee Joo Chng, Boon Cher Goh. Elucidating the synergistic action of histone deacetylase inhibitor and cisplatin in lung squamous cell carcinoma. [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 974. doi:10.1158/1538-7445.AM2013-974

Abstract 2741: Synergistic inhibition of tumor growth and overcoming chemo-resistance by simultaneously targeting key components in DNA damage/repair, epigenetic, and putative cancer stem cell signaling pathways using novel dual-functional DNA-alkylating/HDAC inhibitor and tumor suppressor gene nanoparticles in lung cancer

Abstract In this study we explored a novel therapeutic strategy aimed to simultaneously target multiple key components in DNA damage/repair, epigenetic, and putative cancer stem cell (CSC) signaling pathways using a novel dual-target DNA-alkylating nitrogen mustard/HDAC inhibitor (DANM/HDACi) and tumor suppressor gene (TSG) nanoparticles to promote antitumor synergy and overcome drug resistance in lung cancer. We evaluated therapeutic effects of a new class DNAM/HDACi, NL10, as a single agent or in combination with novel TSGs NPRL2, a regulator of the DNA damage checkpoint pathway, and p53, a regulator of apoptosis and drug resistance in the DNA damage/repair pathway in more than 50 human NSCLC and SCLC cell lines. We detected a high anti-tumor potency by NL101 treatment across lung cancer cell lines, with IC50 doses ranging from nanomolar to lower micromolar levels. A 20 to 100-fold higher level of cytotoxicity was observed across these cell lines, particularly in SCLC cell lines, treated by NL101 than in those treated by bendamustine, a nitrogen mustard that is currently in Phase II/III clinical trials in SCLC patients. We analyzed the NL101-induced DNA damage in NSCLC H1299 cells and found that NL101 induced a significantly higher degree of DNA damage than that induced by either bendamustine or cisplatin at the same IC20 dose level for each agent. We analyzed the DNA-damage-induced apoptosis in NSCLC cells treated by NL101 alone or in combination with DOTAP:Cholesterol (DC)-complexed NPRL2- or p53-expressing plasmid DNA nanoparticles. We detected a significantly enhanced apoptosis in both combination treatments with NL101+DC-NPRL2 and NL101+DC-p53. We also found that combination treatment with NL101 and DC-NPRL2 nanoparticles promoted a synergistic inhibitory effect on tumor cell-induced clonogenecity in H1299 cells and on tumor cell growth in SCLC H69 and GLC16 cells at effective dose levels of NL101 from IC10 to IC75. Furthermore, we found that the sensitivity of lung cancer cells to NL101 treatment showed a significantly negative correlation with expression levels of the HDAC family proteins HDAC1, HDAC2, HDAC3, and HDAC5 and the putative lung cancer stem cell (CSC) marker ALDH1 protein. We found a dose-dependent inhibition of multiple HDACs and CSC markers in both NSCLC and SCLC cells. Our results suggest that a combination treatment using a novel DANM/HDACi with pro-apoptotic TSGs targeting the DNA damage/repair, epigenetic, and CSC signaling pathways will enhance chemotherapeutic sensitivity, promote anti-cancer synergism, overcome drug resistance, and block tumor progression and relapse by targeting putative CSCs. (Supported by grants P50CA70907, RO1 CA-116322, and W81XWH0920139) 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 2741. doi:1538-7445.AM2012-2741

Abstract 870: Synergistic antitumor activity of AKT inhibitor MK2206 and FUS1-nanoparticles in LKB1 mutant NSCLC

Abstract FUS1, a novel tumor suppressor gene in the human chromosome 3p21.3 region, is deleted in many cancers. A phase I clinical trial assessing FUS1-mediated molecular therapy has reported antitumor activity in lung cancer patients. Previous studies showed that FUS1 regulates the activation of multiple oncogenic kinases. MK2206 is a highly selective non-ATP-competitive allosteric inhibitor of AKT currently being evaluated in early-phase clinical trials for treatment of patients with lung cancer. In this study, we evaluated the combined effects of the tumor suppressor gene FUS1 and MK2206 on tumor cell growth and apoptosis induction in NSCLC cells and explored the molecular mechanism of their mutual action. We found that exogenous expression of FUS1 sensitized the response of NSCLC cells to MK2206, resulting in a marked increase in growth suppression and apoptosis in LKB1-mutant NSCLC cells. However, FUS1 had no effect on the response of LKB1-wild-type NSCLC cells to MK2206. Systemic treatment with a combination of FUS1-nanoparticles and MK2206 in an LKB1 mutant H322 lung cancer subcutaneous xenograft mouse model enhanced the therapeutic efficacy of MK2206. The mice receiving the combination of MK2206 and FUS1-nanoparticles showed a significantly reduced mean tumor volume (364±164 mm3) compared with mice receiving empty vector/MK2206 (728±101 mm3), MK2206 alone (926±250 mm3), FUS1 alone (1171±294 mm3), or empty vector (2405±737 mm3) by day 21 (P<0.01 for all three comparisons). Our results show that FUS1 may play a role in modulating the sensitivity of lung cancer cells to AKT inhibition, and suggests that a combination of FUS1-nanoparticles and MK2206 may be an effective treatment strategy for LKB1 mutant human lung cancers. 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 870. doi:1538-7445.AM2012-870

Abstract LB-390: Antitumor efficacy of crizotinib (PF-02341066), a potent and selective ALK and c-Met RTK inhibitor, in EML4-ALK driven NSCLC tumors in vitro and in vivo

Abstract EML4-ALK fusion was recently characterized as an “Addicted Oncogene” in a subset of human lung adenocarcinoma, and it plays an essential role in regulation of the tumor cell survival, growth and metastasis. Crizotinib is a potent and selective ATP competitive small molecule inhibitor of ALK and c-Met. It is currently in clinical trials for advanced non-small cell lung cancers positive for ALK fusion. This report summarizes the pre-clinical pharmacology studies for crizotinib in the EML4-Alk positive tumor models to assess the pharmacodynamic inhibition of EML4-ALK, antitumor efficacy, PKPD relationships and antitumor mechanism of action in vitro and in vivo. Crizotinib potently inhibited the catalytic activity of ALK kinase (Ki = 0.5 nM) and the autophosphorylation of cellular EML4-ALK V1, V2, V3a and V3b with IC50 values ranging from 26–74 nM. Crizotinib also inhibited cell proliferation and induced apoptosis in NCI-H3122 human NSCLC cells harboring EML4-ALK V1 fusion with IC50s of 63 nM and 110 nM respectively. In the EML4-ALK V3a/b positive NCI-H2228 human NSCLC cells, crizotinib completely inhibited ALK phosphorylation (IC50 = 74 nM) but only partial inhibited cell proliferation and survival. This observation is consistent with the notion that only a portion of H2228 cells are EML4-ALK positive by Exon Array analysis. Crizotinib demonstrated marked tumor growth inhibition and regression in H3122 xenograft model at well tolerated dose levels. The antitumor efficacy of crizotinib was dose dependent and demonstrated a strong correlation to pharmacodynamic inhibition of ALK phosphorylation in vivo. PKPD modeling was conducted to understand the relationships between crizotinib plasma concentration to ALK target inhibition (EC50 = 19 nM) and antitumor efficacy (EC50 = 23 nM). Collectively, the results from H3122 model indicated that significant inhibition of EML4-ALK during the entire treatment period was necessary to achieve robust antitumor efficacy. Additional in vivo studies with crizotinib demonstrated dose dependent inhibition of EML4-ALK mediated signal transduction (STAT3, AKT, Erk, PLCγ, c-Myc), tumor cell proliferation (Ki67) and induction of apoptosis (caspase-3). Crizotinib also dose dependently inhibited total EML4-ALK levels in H3122 tumors indicating an additional antitumor mechanism of action by crizotinib in these tumors. Furthermore, a dose dependent increase in phospho-EGFR levels was observed after 4 days of drug treatment in H3122 model, indicating a compensation mechanism of “oncogene switching” in tumor cell signaling, and a potential resistance mechanism that may compromise “patient” responses to crizotinib treatment in these tumors. In conclusion, crizotinib was shown to be a potent inhibitor of EML4-ALK. It demonstrated marked antitumor efficacy in EML4-ALK dependent human NSCLC tumors in vitro and in vivo. 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 LB-390. doi:10.1158/1538-7445.AM2011-LB-390

343 Targeting p21-activated kinase 1 (PAK1) to induce apoptosis of squamous NSCLC cells

343 Targeting p21-activated kinase 1 (PAK1) to induce apoptosis of squamous NSCLC cells

Adenovirus-mediated SOCS3 gene transfer inhibits the growth and enhances the radiosensitivity of human non-small cell lung cancer cells

The Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway is one of the most important components of cytokine signaling cascades. JAK-STAT signaling pathway modulates various fundamental biological processes and cancer pathogenesis. JAK-STAT is controlled by negative regulators that include suppressors of cytokine signaling (SOCS) proteins. Failure of feedback suppression by SOCS proteins may result in activated JAK-STAT signaling. Methylation-mediated silencing of SOCS3 has been reported in non-small lung cancer (NSCLC) and other human cancers. In this study, we restored SOCS3 expression using adenovirus-mediated gene transfer in NSCLC cells. Infection with a SOCS3-expressing vector inhibited the growth of lung cancer cells, with or without SOCS3 expression, at 2-3 days after infection. The growth inhibition of lung cancer cells was associated with suppressing entry into the S-phase. Restoration of SOCS3 expression induced apoptosis of NSCLC cells that did not express SOCS3. In addition, overexpression of SOCS3 by adenoviral transfer enhanced the radiosensitivity of treated NSCLC cells. In conclusion, our findings may provide insights into the development of applications of SOCS3 gene therapy for lung cancer and, possibly, other human cancers.

In vitro and in vivo characterisation of a novel c-FLIP-targeted antisense phosphorothioate oligonucleotide

Previous studies have suggested that the caspase 8 inhibitor FLIP is a promising anti-cancer therapeutic target. In this study, we characterised a novel FLIP-targeted antisense phosphorothioate oligonucleotide (AS PTO). FLIP AS and control PTOs were assessed in vitro in transient transfection experiments and in vivo using xenograft models in Balb/c nude mice. FLIP expression was assessed by QPCR and Western. Apoptosis induction was determined by flow cytometry and Western. Of 5 sequences generated, one potently down-regulated FLIP. AS PTO-mediated down-regulation of FLIP resulted in caspase 8 activation and apoptosis induction in non-small cell lung (NSCLC) cells but not in normal lung cells. Similar results were observed in colorectal and prostate cancer cells. Furthermore, the FLIP AS PTO sensitized cancer cells but not normal lung cells to apoptosis induced by rTRAIL. Moreover, the FLIP AS PTO enhanced chemotherapy-induced apoptosis in NSCLC cells. Importantly, compared to a control non-targeted PTO, intra-peritoneal delivery of FLIP AS PTO inhibited the growth of NSCLC xenografts and enhanced the in vivo antitumour effects of cisplatin. We have identified a novel FLIP-targeted AS PTO that has in vitro and in vivo activity and which therefore has potential for further pre-clinical development.

A Novel Mono‐carbonyl Analogue of Curcumin Induces Apoptosis by Activating ER Stress in Non‐Small Lung Cancer Cells

Non‐small lung cancer (NSCLC) is one of the most common cancers worldwide. Curcumin, a well‐known food additive and constituent of traditional medicine, has been used to treat several different cancers including NSCLC in clinical trials. However, its poor bioavailability and pharmacokinetic profiles due to its instability under physiological conditions have limited its application in anti‐cancer therapies. In this study, we synthesized a novel mono‐carbonyl analogue of curcumin, (1E,4E)‐1,5‐bis (2‐methoxyphenyl) penta‐1,4‐dien‐3‐one (B63), and examined its proapoptotic effect in NSCLC cells.MethodsH460 or H358 cells were treated with different amounts of curcumin and B63 for various time periods. Cell proliferation and apoptosis were measured by MTT assay and flow cytometry. The activation of ER stress was determined by Western blot analysis. The ER Ca2+ content was measured using the fluorescent Ca2+ indicator fura 2/AM. Lentiviral shRNA was used to knock down the ER stress responsive gene CHOP.ResultsB63 had better bioavailability and more potent proapoptotic activity than curcumin. B63, not curcumin, significantly induced depletion of ER Ca2+ store and activation of the unfolded protein response (UPR). Downregulation of CHOP expression inhibited B63‐induced apoptosis.ConclusionActivation of the UPR represents a key molecular mechanism underlying B63‐induced apoptosis in NSCLC cells.

Unphosphorylated STAT6 contributes to constitutive cyclooxygenase-2 expression in human non-small cell lung cancer

Cyclooxygenase-2 (COX-2) is frequently overexpressed in human cancers and contributes to the malignant phenotype. Our data indicate unphosphorylated signal transducers and activators of transcription 6 (STAT6) may transcriptionally upregulate COX-2 expression and protect against apoptosis in NSCLC cells. In A427 and H2122, NSCLC cell lines that constitutively express COX-2, only unphosphorylated STAT6 was detectable by western blot, thus, all of the following STAT6-dependent effects are attributed to the unphosphorylated protein. In both cell lines, small-interfering RNA-mediated knockdown of STAT6 or stable expression of dominant-negative STAT6 decreased COX-2 expression. In contrast, transfection with a phosphorylation-deficient mutant STAT6 increased COX-2 levels. Immunofluorescent staining revealed the presence of STAT6 in H2122 nuclei, suggesting a direct role in gene regulation for the unphosphorylated protein. Consistent with this hypothesis, unphosphorylated STAT6 increased luciferase expression from a COX-2 promoter reporter construct. STAT6 co-immunoprecipitated with the transcriptional co-activator, p300, and chromatin immunoprecipitation assays demonstrated that these proteins bind a consensus STAT6 binding site located within the COX-2 promoter. STAT6 DNA-binding specificity was confirmed by electrophoretic mobility shift assay. As COX-2 over-expression has been clearly linked to apoptosis resistance and other hallmarks of malignancy, these findings suggest a novel role of unphosphorylated STAT6 in the pathogenesis of non-small cell lung cancer.

Release of bFGF following apoptosis and necrosis in NSCLC cells: effects on chemosensitivity to cisplatin

The majority of NSCLC cell lines produce elevated levels of bFGF but do not secrete considerable amounts. We therefore investigated the influence of cell death inducing mechanisms on bFGF release. In addition we studied the biological consequences on chemosensitivity of NSCLC cell lines to cisplatin. In our experiment we induced i) apoptosis with cisplatin and ii) necrosis with heat treatment in NSCLC cell lines. Supernatants of apoptotic/necrotic NSCLC cell lines exhibited significantly increased levels of bFGF independent of the cell death inducing mechanism. The incubation of three NSCLC cell lines with these supernatants resulted in enhanced cisplatin-induced apoptosis/necrosis in one of the cell lines (H1299) only. Addition of an anti-bFGF antibody to conditioned supernatant of H1299 cells abolished this effect partially. These results suggest that the release of bFGF following cell death contributes to enhanced chemosensitivity in NSCLC cell lines.

Curcumin enhances Vinorelbine mediated apoptosis in NSCLC cells by the mitochondrial pathway

Elderly lung cancer patients and those with poor performance status/co-morbid conditions are deprived of chemotherapy because of high toxicity of multidrug regimens. Human squamous cell lung carcinoma H520 cells treated with Curcumin were sensitized to the cytotoxicity caused by chemotherapeutic agent, Vinorelbine. Both caused apoptosis by increasing the protein expression of Bax and Bcl-xs while decreasing Bcl-2 and Bcl-X L, releasing apoptogenic cytochrome c, and augmenting the activity of caspase-9 and caspase-3. Expression of Cox-2, NF-κB, and AP-1 was also affected. 23.7% apoptosis was induced in the H520 cells by treatment with Curcumin while Vinorelbine caused 38% apoptosis. Pre-treatment with Curcumin enhanced the Vinorelbine induced apoptosis to 61.3%. The findings suggest that Curcumin has the potential to act as an adjuvant chemotherapeutic agent and enhance chemotherapeutic efficacy of Vinorelbine in H520 cells in vitro. Thus, Curcumin offers the prospect of being beneficial in the above-mentioned patient groups.

CACNA2D2-mediated apoptosis in NSCLC cells is associated with alterations of the intracellular calcium signaling and disruption of mitochondria membrane integrity.

The CACNA2D2 gene, a new subunit of the Ca(2+)-channel complex, was identified in the homozygous deletion region of chromosome 3p21.3 in human lung and breast cancers. Expression deficiency of the CACNA2D2 in cancer cells suggests a possible link of it to Ca(2+) signaling in the pathogenesis of lung cancer and other cancers. We investigated the effects of overexpression of CACNA2D2 on intracellular Ca(2+) contents, mitochondria homeostasis, cell proliferation, and apoptosis by adenoviral vector-mediated wild-type CACNA2D2 gene transfer in 3p21.3-deficient nonsmall cell lung cancer cell lines. Exogenous expression of CACNA2D2 significantly inhibited tumor cell growth compared with the controls. Overexpression of CACNA2D2 induced apoptosis in H1299 (12.5%), H358 (13.7%), H460 (22.3%), and A549 (50.1%) cell lines. Levels of intracellular free Ca(2+) were elevated in AdCACNA2D2-transduced cells compared with the controls. Mitochondria membrane depolarization was observed prior to apoptosis in Ad-CACNA2D2 and Adp53-transduced H460 and A549 cells. Release of cyt c into the cytosol, caspase 3 activation, and PARP cleavage were also detected in these cells. Together, these results suggest that one of the pathways in CACNA2D2-induced apoptosis is mediated through disruption of mitochondria membrane integrity, the release of cyt c, and the activation of caspases, a process that is associated with regulation of cytosolic free Ca(2+) contents.

CD 437 enhances TRAIL-induced apoptosis in NSCLC cells

CD 437 enhances TRAIL-induced apoptosis in NSCLC cells