Chuanpit Ninsontia

Silymarin selectively protects human renal cells from cisplatin-induced cell death

Context: Cisplatin-induced nephrotoxicity has been accepted as an important obstacle for efficient cisplatin-based chemotherapy. Silymarin from seeds of milk thistle [Silybum marianum L. (Asteraceae)] has been shown to possess various potential pharmacological properties; however, whether or not this agent selectively protects renal cells from cisplatin-induced cell death with no interfering effect on cancer cells is not clear. Objective: Potential of silymarin in protection of cisplatin-induced renal cell death without compromising effect on anticancer activity of cisplatin was demonstrated in this study. Materials and methods: Cisplatin-induced cell death was evaluated in human proximal tubular HK-2, lung carcinoma H460, and melanoma G361 cells using MTT, Hoechst 33342, and propidium iodide assays. Results: Cisplatin induced both apoptosis and necrosis in HK-2 cells and caused a decrease in cell viability by ~40% and 60% at the doses of 25 and 100 µM, respectively. Pretreatment with 25–200 µM of silymarin significantly protected against cisplatin-induced cell death in a dose-dependent manner. In contrast, pretreatment of silymarin (25–100 µM) caused no significant change on cisplatin-induced cell death in H460 cells but significantly potentiated cisplatin-induced apoptosis in G361 cells. Discussion and conclusion: These findings reveal the selectivity of silymarin in protection of renal cells from cisplatin-induced cell death and could be beneficial for the development of this considerately safe compound as a renoprotective agent.

Zinc induces epithelial to mesenchymal transition in human lung cancer H460 cells via superoxide anion-dependent mechanism

BackgroundEpithelial to mesenchymal transition (EMT) has been shown to be a crucial enhancing mechanism in the process of cancer metastasis, as it increases cancer cell capabilities to migrate, invade and survive in circulating systems. This study aimed to investigate the effect of essential element zinc on EMT characteristics in lung cancer cells.MethodsThe effect of zinc on EMT was evaluated by determining the EMT behaviors using migration, invasion and colony formation assay. EMT markers were examined by western blot analysis. Reactive oxygen species (ROS) were detected by specific fluorescence dyes and flow cytometry. All results were analyzed by ANOVA, followed by individual comparisons with post hoc test.ResultsThe present study has revealed for the first time that the zinc could induce EMT and related metastatic behaviors in lung cancer cells. Results showed that treatment of the cells with zinc resulted in the significant increase of EMT markers N-cadherin, vimentin, snail and slug and decrease of E-cadherin proteins. Zinc-treated cells exhibited the mesenchymal-like morphology and increased cancer cell motility with significant increase of activated FAK, Rac1, and RhoA. Also, tumorigenic abilities of lung cancer cells could be enhanced by zinc. Importantly, the underlying mechanism was found to be caused by the ability of zinc to generate intracellular superoxide anion. Zinc was shown to induce cellular superoxide anion generation and the up-regulation of EMT markers and the induced cell migration and invasion in zinc-treated cells could be attenuated by the treatment of MnTBAP, a specific superoxide anion inhibitor.ConclusionKnowledge gains from this study may highlight the roles of this important element in the regulation of EMT and cancer metastasis and fulfill the understanding in the area of cancer cell biology.

Benzophenone-3 increases metastasis potential in lung cancer cells via epithelial to mesenchymal transition

Exposure to compounds with cancer-potentiating effects can contribute to the progression of cancer. Herein we have discovered for the first time that benzophenone-3 (BP-3), a chemical used as sunscreen in various cosmetic products, enhances the ability of lung cancer cells to undergo metastasis. The exposure of the lung cancer cells to BP-3 at non-toxic concentrations significantly increased the number of anoikis resistant cells in a dose-dependent manner. Also, BP-3 increased the growth rate as well as the number of colonies accessed by anchorage-independent growth assay. We found that the underlying mechanisms of such behaviors were the epithelial to mesenchymal transition (EMT) process of cancer cells, and the increase in caveolin-1 (Cav-1) expression. As both mechanistic events mediated anoikis resistance via augmentation of cellular survival signals, our results further revealed that the BP-3 treatment significantly up-regulated extracellular-signal-regulated kinase (ERK). Also, such compounds increased the cellular levels of anti-apoptotic Bcl-2 and Mcl-1 proteins. As the presence of a substantial level of BP-3 in plasma of the consumers has been reported, this finding may facilitate further investigations that lead to better understanding and evidence concerning the safety of use in cancer patients.

Zinc suppresses stem cell properties of lung cancer cells through protein kinase C-mediated β-catenin degradation

Highly tumorigenic cancer stem cells (CSCs) residing in most cancers are responsible for cancer progression and treatment failure. Zinc is an element regulator of several cell functions; however, its role in regulation of stem cell program in lung cancer has not been demonstrated. The present study reveals for the first time that zinc can suppress stem cell properties of lung cancer cells. Such findings were proved in different lung cancer cell lines (H460, H23, and H292) and it was found that CSC markers (CD133 and ALDH1A1), stem cell-associated transcription factors (Oct4, Nanog, and Sox-2), and the ability to form tumor spheroid were dramatically suppressed by zinc treatments. Zinc was found to activate protein kinase C-α (PKCα) that further phosphorylated and mediated β-catenin degradation through the ubiquitin-proteasomal pathway. Zinc was found to increase the β-catenin-ubiquitin complex, which can be inhibited by a specific PKC inhibitor, bisindolylmaleimide I. Using specific reactive oxygen species detection and antioxidants, we have demonstrated that superoxide anions generated by zinc are a key upstream mechanism for PKCα activation leading to the subsequent suppression of stem cell features of lung cancer. Zinc increased cellular superoxide anions and the addition of superoxide anion scavenger prevented the activation of PKCα and β-catenin degradation. These findings indicate a novel role for zinc regulation in the PKCα/β-catenin pathway and explain an important mechanism for controlling of stem cell program in lung cancer cells.

Cytoplasmic p21 Mediates 5-Fluorouracil Resistance by Inhibiting Pro-Apoptotic Chk2

The oncogenic cytoplasmic p21 contributes to cancer aggressiveness and chemotherapeutic failure. However, the molecular mechanisms remain obscure. Here, we show for the first time that cytoplasmic p21 mediates 5-Fluorouracil (5FU) resistance by shuttling p-Chk2 out of the nucleus to protect the tumor cells from its pro-apoptotic functions. We observed that cytoplasmic p21 levels were up-regulated in 5FU-resistant colorectal cancer cells in vitro and the in vivo Chorioallantoic membrane (CAM) model. Kinase array analysis revealed that p-Chk2 is a key target of cytoplasmic p21. Importantly, cytoplasmic form of p21 mediated by p21T145D transfection diminished p-Chk2-mediated activation of E2F1 and apoptosis induction. Co-immunoprecipitation, immunofluorescence, and proximity ligation assay showed that p21 forms a complex with p-Chk2 under 5FU exposure. Using in silico computer modeling, we suggest that the p21/p-Chk2 interaction hindered the nuclear localization signal of p-Chk2, and therefore, the complex is exported out of the nucleus. These findings unravel a novel mechanism regarding an oncogenic role of p21 in regulation of resistance to 5FU-based chemotherapy. We suggest a possible value of cytoplasmic p21 as a prognosis marker and a therapeutic target in colorectal cancer patients.

Abstract 1904: Novel 5-fluorouracil-thymoquinone hybrid kills colon cancer stem cells

Objectives: Recently, it has been shown that a subset population of cancer cells in a tumor, the so-called cancer stem cells (CSCs), majorly contributes to chemoresistance and the ability of a tumor to form recurrences and metastases. Since the response to the standard chemotherapeutic drug 5-fluorouracil (5-FU) in patients with colorectal cancer (CRC) leads only to an inadequate response, we aimed to synthesize new hybrid molecules by combining 5-FU with the plant-derived drug thymoquinone (TQ). Methods: The cytotoxic effect of hybrids, the single drugs and the combination was investigated by crystal violet assays. HCT116, HT29 and 5-FU-resistant colorectal tumor cells were used to assess the spheroid forming capability after treatment with different concentrations of the hybrid and the single drugs by staining with the fluorescent stem cell marker CDy1 and Hoechst 3324 after 21 days of incubation. Western Blot analysis for stem cell surface marker CD133 were performed to verify these results. We have analyzed the anticancer effects of the single drugs TQ and 5-FU and the most effective hybrid on deregulation of cancer signaling pathways utilizing the NANOSTRING cancer pathway panel. In addition, we performed a single cell cloning from spheroids to generate monoclonal cell lines with enriched CSC populations. In vivo effects were studied in the chick chorioallantoic membrane (CAM) assay and in mouse xenografts. Results: The 5FU-TQ hybrid has an increased cytotoxic effect towards CRC cells when compared to single drug treatments. The spheroid formation assay revealed the capability of the hybrid to reduce sphere number and size at fairly low concentrations (10, 20 µM), however at 50 µM there was a complete suppression of spheroid growth. NANOSTRING analysis showed that gene expression of main components of the PI3K/AKT signaling pathway was significantly down-regulated after treatment with all drugs but most prominently with the new hybrid. After data processing we found that AKT3, IGF1, COL11A1, FGF10, GNG7, EPO genes were downregulated in hybrid-treated cells, while CDKN1A, BAX, JUN, MDM2, FAS were upregulated. This was verified via Western Blotting for a panel of markers. Next, we investigated the effectiveness of the hybrid to kill clones having an enriched CSC population. Indeed, CD133 protein levels were significantly decreased in hybrid-treated clones. Histological analysis of in vivo CAM grafts showed that HCT116 control cells developed a well vascularized tumor mass whereas HCT116 cells that were treated with the hybrid showed no more vital tumor cells, but extended areas of necrosis, calcification, and desmoplasia. In vivo mouse xenografts verified the potential of this hybrid as an antitumor agent reducing tumor volume significantly. Conclusion: Our findings strongly suggest that the newly synthesized 5-FU-TQ hybrid might become a promising anticancer drug by directly targeting CSCs. Citation Format: Benardina Ndreshkjana, Aysun Capci Karagoz, Volker Klein, Pithi Chanvorachote, Julienne K. Muenzner, Kerstin Huebner, Chuanpit Ninsontia, Sara Steinmann, Abbas Agaimy, Maamoun Fatfat, Hala Gali-Muhtasib, Svetlana B. Tsogoeva, Regine Schneider-Stock. Novel 5-fluorouracil-thymoquinone hybrid kills colon cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1904. doi:10.1158/1538-7445.AM2017-1904