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Combination of tolfenamic acid and curcumin induces colon cancer cell growth inhibition through modulating specific transcription factors and reactive oxygen species.

ABSTRACT: Curcumin (Cur) has been extensively studied in several types of malignancies including colorectal cancer (CRC); however its clinical application is greatly affected by low bioavailability. Several strategies to improve the therapeutic response of Cur are being pursued, including its combination with small molecules and drugs. We investigated the therapeutic efficacy of Cur in combination with the small molecule tolfenamic acid (TA) in CRC cell lines. TA has been shown to inhibit the growth of human cancer cells in vitro and in vivo, via targeting the transcription factor specificity protein1 (Sp1) and suppressing survivin expression. CRC cell lines HCT116 and HT29 were treated with TA and/or Cur and cell viability was measured 24-72 hours post-treatment. While both agents caused a steady reduction in cell viability, following a clear dose/ time-dependent response, the combination of TA+Cur showed higher growth inhibition when compared to either single agent. Effects on apoptosis were determined using flow cytometry (JC-1 staining to measure mitochondrial membrane potential), Western blot analysis (c-PARP expression) and caspase 3/7 activity. Reactive oxygen species (ROS) levels were measured by flow cytometry and the translocation of NF-kB into the nucleus was determined using immunofluorescence. Results showed that apoptotic markers and ROS activity were significantly upregulated following combination treatment, when compared to the individual agents. This was accompanied by decreased expression of Sp1, survivin and NF-kB translocation. The combination of TA+Cur was more effective in HCT116 cells than HT29 cells. These results demonstrate that TA may enhance the anti-proliferative efficacy of Cur in CRC cells.

SUBMITTER: Sankpal UT

PROVIDER: S-EPMC4823099 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundCurcumin inhibits growth of several cancer cell lines, and studies in this laboratory in bladder and pancreatic cancer cells show that curcumin downregulates specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and pro-oncogenic Sp-regulated genes. In this study, we investigated the anticancer activity of curcumin and several synthetic cyclohexanone and piperidine analogs in colon cancer cells.MethodsThe effects of curcumin and synthetic analogs on colon cancer cell proliferation and apoptosis were determined using standardized assays. The changes in Sp proteins and Sp-regulated gene products were analysed by western blots, and real time PCR was used to determine microRNA-27a (miR-27a), miR-20a, miR-17-5p and ZBTB10 and ZBTB4 mRNA expression.ResultsThe IC50 (half-maximal) values for growth inhibition (24 hr) of colon cancer cells by curcumin and synthetic cyclohexanone and piperidine analogs of curcumin varied from 10 ?M for curcumin to 0.7 ?M for the most active synthetic piperidine analog RL197, which was used along with curcumin as model agents in this study. Curcumin and RL197 inhibited RKO and SW480 colon cancer cell growth and induced apoptosis, and this was accompanied by downregulation of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and Sp-regulated genes including the epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor (c-MET), survivin, bcl-2, cyclin D1 and NF?B (p65 and p50). Curcumin and RL197 also induced reactive oxygen species (ROS), and cotreatment with the antioxidant glutathione significantly attenuated curcumin- and RL197-induced growth inhibition and downregulation of Sp1, Sp3, Sp4 and Sp-regulated genes. The mechanism of curcumin-/RL197-induced repression of Sp transcription factors was ROS-dependent and due to induction of the Sp repressors ZBTB10 and ZBTB4 and downregulation of microRNAs (miR)-27a, miR-20a and miR-17-5p that regulate these repressors.ConclusionsThese results identify a new and highly potent curcumin derivative and demonstrate that in cells where curcumin and RL197 induce ROS, an important underlying mechanism of action involves perturbation of miR-ZBTB10/ZBTB4, resulting in the induction of these repressors which downregulate Sp transcription factors and Sp-regulated genes.

Project description:Purpose: Despite considerable efforts to improve treatment modalities for cholangiocarcinoma, a common form of malignant tumor, its long-term survival rate remains poor. Hydroxychloroquine (HCQ) is a 4-aminoquinoline derivative antimalarial drug that has antimalarial and autophagy inhibition effects and exhibits comprehensive therapeutic effects on various cancers. In this study, we aimed to explore the anticancer potential and the underlying molecular mechanism of HCQ in cholangiocarcinoma treatment in vitro and in vivo. Methods: Autophagy-related genes (ARGs) were obtained from the Human Autophagy Database and Molecular Signatures Database, and the expression profiles of ARGs were downloaded from the database of The Cancer Genome Atlas. Different expression gene sets were performed using R software. The Gene Ontology and KEGG enrichment analyses were performed to reveal significantly enriched signaling pathways and to identify differentially expressed genes in cholangiocarcinoma tissues. HuCCT-1 and CCLP-1 cells were exposed to different concentrations of HCQ. Cell proliferation was detected by Cell Counting Kit-8 (CCK-8), colony formation, and 5-ethynyl-2'-deoxyuridine (EdU) assays. Cell apoptosis and cycle arrest were detected by the Live/Dead cell assay and flow cytometry (FCM). The inhibition of autophagy was observed using fluorescence microscopy. The reactive oxygen species levels were assessed by fluorescence microscopy and flow cytometry. The protein levels were determined by western blot. A cholangiocarcinoma cell line xenograft model was used to evaluate the antitumor activity of HCQ in vivo. Results: Compared with normal tissues, there were 141 ARGs with an aberrant expression in cholangiocarcinoma tissues which were mainly enriched in autophagy-related processes. Inhibition of autophagy by HCQ effectively suppressed cholangiocarcinoma in vitro and in vivo. HCQ inhibited cell proliferation and induced apoptosis and cycle arrest in vitro by increasing ROS accumulation, which was involved in autophagy inhibition. The ROS scavenger reduced l-glutathione distinctly weakened HCQ-induced cell apoptosis and viability inhibition in cholangiocarcinoma cells. In addition, HCQ inhibited growth of cholangiocarcinoma cell line xenograft tumors. Conclusion: HCQ could inhibit cell proliferation and induce apoptosis in cholangiocarcinoma by triggering ROS accumulation via autophagy inhibition, which makes HCQ a potential antitumor drug candidate for cholangiocarcinoma treatment.

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Combination of tolfenamic acid and curcumin induces colon cancer cell growth inhibition through modulating specific transcription factors and reactive oxygen species.

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