Cytochalasan Alkaloids as TRAIL Sensitizers from an Endophytic Fungus Chaetomium sp.

Two new cytochalasans with a rare 6/6/5/5/7 pentacyclic ring system, named chaetoconvosins C−D (1−2), together with two known congeners (3−4), were isolated from the fermentation of an endophytic fungus, Chaetomium sp. SG-01, harbored in the fibrous roots of Schisandra glaucescens Diels. Their structures including the absolute configuration were elucidated by extensive spectroscopic (HRESIMS, NMR, and ECD) and X-ray crystallographic analyses. The TRAIL sensitivity of 1–4 in a TRAIL-resistant HT29 colorectal cancer cell line was evaluated, which revealed that co-treatment of 1–4 at 50 µM with TRAIL (150 ng/mL) reduced the HT29 cell viability by 19.0%, 24.1%, 17.9%, and 15.5%, respectively, compared to treatment with 1–4 alone.

The combined effect of dichloroacetate and 3-bromopyruvate on glucose metabolism in colorectal cancer cell line, HT-29; the mitochondrial pathway apoptosis

Background 5-Fluorouracil (5-FU) is regarded as the first line treatment for colorectal cancer; however, its effectiveness is limited by drug resistance. The ultimate goal of cancer therapy is induction of cancer cell death to achieve an effective outcome with minimal side effects. The present work aimed to assess the anti-cancer activities of mitocans which can be considered as an effective anticancer drug due to high specificity in targeting cancer cells. Methods MTT (3–4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) assay was performed to determine the effects of our mitocans on cell viability and cell death. Apoptosis and necrosis, caspase 3 activity, mitochondrial membrane potential and ROS production in HT29 cell lines were analyzed by ApopNexin™ FITC/PI Kit, Caspase- 3 Assay Kit, MitoTracker Green and DCFH-DA, respectively. Moreover, quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression level of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) genes in HT29 cell lines. Results Treatment with mitocans (3Br-P + DCA) inhibited the growth of HT29. Moreover, 3Br-P + DCA significantly induced apoptosis and necrosis, activation of caspase 3 activity, depolarize the mitochondrial membrane potential, and ROS production. At a molecular level, 3Br-P + DCA treatment remarkably down-regulated the expression of Bcl-2, while up-regulated the expression of Bax. Conclusion Mitocans, in particular the combined drug, 3Br-P + DCA, could be regarded and more evaluated as a safe and effective compound for CRC treatment. Targeting hexokinase and pyruvate dehydrogenase kinase enzymes may be an option to overcome 5-FU -mediated chemo-resistant in colorectal cancer.

CYTOTOXICITY EFFECT OF IONIC LIQUID-GRAVIOLA FRUIT (ANNONA MURICATA) EXTRACT TO HUMAN COLON CANCER (HT29) CELL LINES

The present study aimed to investigate the anti-proliferative effect of the ionic liquid-Graviola fruit (IL-GFE) extract on colon adenocarcinoma (HT29) cell lines and their kinetics behaviour to assess the Graviola fruit potential as a therapeutic alternative in cancer treatment. The phytoconstituents content of IL-GFE was identified using GC-TOFMS apparatus and measured its cytotoxicity on HT29 by tetrazolium bromide. Then the cytokinetic behaviour of the treated HT29 cells with IL-GFE was illustrated using the cells' growth curve. Besides, the cell cycle phase perturbation for the treated HT29 was applied using a flow cytometry technique. Qualitative identification of phytoconstituents of IL-GFE showed that Graviola fruit contains acetogenins, alkaloids, flavonoids, tannins and saponins compounds. IL-GF extract displayed a cytotoxicity effect on HT29 cells with the IC50 value of 10.56 µg/mL, while Taxol showed an IC50 value of 1.22 µg/mL. IL-GFE also decreased the cell generation number from 3.93 to 2.96 generations compared to Taxol-treated cells 2.01 generations. The microscope observation of the HT29 cells treated with the crude IL-GFE displayed loss of density and cell detachment. The extract's growth inhibition was related to the cell cycle arrest at the G0/G1 phase. IL-GFE inhibited colon adenocarcinoma HT29 cells' proliferation and affected their kinetic behaviour by lowering cell viability, inducing apoptosis, and arresting the cell cycle at the G0/G1 phase. ABSTRAK: Kajian ini bertujuan untuk mengkaji kesan anti-proliferatif ekstrak buah-ion Graviola (IL-GFE) pada garis sel adenokarsinoma kolon (HT29) dan tingkah laku kinetik mereka untuk menilai potensi buah Graviola sebagai alternatif terapi untuk barah rawatan. Kandungan fitokonstituen IL-GFE dikenal pasti menggunakan alat GC-TOFMS dan mengukur sitotoksisitasnya pada HT29 oleh tetrazolium bromida. Kemudian tingkah laku sitokinetik sel HT29 yang dirawat dengan IL-GFE digambarkan menggunakan keluk pertumbuhan sel. Selain itu, gangguan fasa kitaran sel untuk HT29 yang dirawat diaplikasikan menggunakan teknik sitometri aliran. Pengenalpastian kualitatif fitokonstituen IL-GFE menunjukkan bahawa buah Graviola mengandungi asetogenin, alkaloid, flavonoid, tanin dan sebatian saponin. Ekstrak IL-GF memperlihatkan kesan sitotoksisiti pada sel HT29 dengan nilai IC50 10.56 µg/mL, sementara Taxol menunjukkan nilai IC50 1.22 µg/mL. IL-GFE juga menurunkan jumlah penjanaan sel dari 3.93 hingga 2.96 generasi berbanding sel yang dirawat Taxol 2.01 generasi. Pemerhatian mikroskop sel HT29 yang dirawat dengan IL-GFE kasar menunjukkan kehilangan ketumpatan dan detasmen sel. Perencatan pertumbuhan ekstrak berkaitan dengan penangkapan kitaran sel pada fasa G0/G1. IL-GFE menghalang percambahan sel HT29 adenokarsinoma kolon dan mempengaruhi tingkah laku kinetik mereka dengan menurunkan daya maju sel, mendorong apoptosis, dan menghentikan kitaran sel pada fasa G0/G1.

Study of the effect of Pioglitazone and Cetuximab on Cancer Stem-like Cellsenriched HT29 cell line

One of the major causes of cancer resistance to chemotherapy has been found to be the presence of Cancer Stem Cells (CSCs) in cancerous tissues. Probably, these cells are the source of cancer and the cause of malignancy and recurrence in the affected population. Therefore, it is possible to target CSCs to treat cancer. Since the percentage of CSCs in the total tumor mass is very low, so studies about these cells depend on their isolation and enrichment methods. Some studies have suggested that EMT induction in population of normal epithelial cells and cancer cells by inhibiting of E-cadherin, protects them against chemotherapy, anticancer and apoptosis drugs, moreover they get characteristics of CSCs. So in order to study CSCs can enrich them by inhibiting of E-cadherin in tumor population.In this study, we tried to examine how the effect of Pioglitazone and Cetuximab, two drugs used in chemotherapy of Colon Cancer, on CSCs enriched HT29 cell line (was called HT29-shE) in which CSCs were enriched with induction of EMT by inhibiting of E-cadherin using shRNA.For this purpose, after cell preparation EMT and CSCs markers in Pioglitazone and Cetuximab treated cells were assessed and compared with untreated cells using flow cytometry, real‐time PCR and microscopic monitoring. The findings showed mesenchymal morphology of HT29-shE changed to epithelial morphology after Pioglitazone and Cetuximab treatment, moreover E-cadherin expression increased and Vimentin expression decreased. In addition, expression of CSC markers (CD133+ and CD44+) were reduced in HT29-shE after treatment.

Diclofenac N-Derivatives as Therapeutic Agents with Anti-Inflammatory and Anti-Cancer Effect

A series of diclofenac N-derivatives (2, 4, 6, 8c, 9c, 10a-c) were synthesized in order to test their anti-cancer and anti-inflammatory effects. The anticarcinogen activity has been assayed against three cancer cell lines: HT29, human colon cancer cells; Hep-G2, human hepatic cells; and B16-F10, murine melanoma cells. First, we determined the cytotoxicity of the different compounds, finding that the most effective compound was compound 8c against all cell lines and both compounds 4 and 6 in human Hep-G2 and HT29 cell lines. Compounds 4 and 8c were selected for the percentage of apoptosis determination, cell cycle distribution, and mitochondrial membrane potential measure because these products presented the lowest IC50 values in two of the three cancer cell lines assayed (B16-F10 and HepG2), and were two of the three products with lowest IC50 in HT29 cell line. Moreover, the percentages of apoptosis induction were determined for compounds 4 and 8c, showing that the highest values were between 30 to 60%. Next, the effects of these two compounds were observed on the cellular cycle, resulting in an increase in the cell population in G2/M cell cycle phase after treatment with product 8c, whereas compound 4 increased the cells in phase G0/G1, by possible differentiation process induction. Finally, to determine the possible apoptosis mechanism triggered by these compounds, mitochondrial potential was evaluated, indicating the possible activation of extrinsic apoptotic mechanism. On the other hand, we studied the anti-inflammatory effects of these diclofenac (DCF) derivatives on lipopolysaccharide (LPS) activated RAW 264.7 macrophages-monocytes murine cells by inhibition of nitric oxide (NO) production. As a first step, we determined the cytotoxicity of the synthesized compounds, as well as DCF, against these cells. Then, sub-cytotoxic concentrations were used to determine NO release at different incubation times. The greatest anti-inflammatory effect was observed for products 2, 4, 8c, 10a, 10b, and 9c at 20 µg·mL−1 concentration after 48 h of treatment, with inhibition of produced NO between 60 to 75%, and a concentration that reduces to the 50% the production of NO (IC50 NO) between 2.5 to 25 times lower than that of DCF. In this work, we synthesized and determined for the first time the anti-cancer and anti-inflammatory potential of eight diclofenac N-derivatives. In agreement with the recent evidences suggesting that inflammation may contribute to all states of tumorigenesis, the development of these new derivatives capable of inducing apoptosis and anti-inflammatory effects at very low concentrations represent new effective therapeutic strategies against these diseases.

The Combined Effect of Dichloroacetate and 3-Bromopyruvate in Colorectal Cancer Cell Line, HT-29; The Mitochondrial Pathway Apoptosis

Background 5-Fluorouracil (5-FU) is regarded as the first line treatment for colorectal cancer; however, its effectiveness is limited by drug resistance. The ultimate goal of cancer therapy is induction of cancer cell death to achieve an effective outcome with minimal side effects. The present work aimed to assess the anti-cancer activities of mitocans which can be considered as an effective anticancer drug due to high specificity in targeting cancer cells. Methods MTT (3–4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) assay was performed to determine the effects of our mitocans on cell viability and cell death. Apoptosis and necrosis, caspase 3 activity, mitochondrial membrane potential and ROS production in HT29 cell lines were analyzed by ApopNexin™ FITC/PI Kit, Caspase- 3 Assay Kit, MitoTracker Green and DCFH-DA, respectively. Moreover, quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression level of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) genes in HT29 cell lines. Results treatment with mitocans (3Br-P + DCA) inhibited the growth of HT29. Moreover, 3Br-P + DCA significantly induced apoptosis and necrosis, activation of caspase 3 activity, depolarize the mitochondrial membrane potential, and ROS production. At a molecular level, 3Br-P + DCA treatment remarkably down-regulated the expression of Bcl-2, while up-regulated the expression of Bax. Conclusion mitocans, in particular the combined drug, 3Br-P + DCA, could be regarded and more evaluated as a safe and effective compound for CRC treatment. Targeting hexokinase and pyruvate dehydrogenase kinase enzymes may be an option to overcome 5-FU -mediated chemo-resistant in colorectal cancer.

Synthesis and Characterization of Chitosan-Based Nanodelivery Systems to Enhance the Anticancer Effect of Sorafenib Drug in Hepatocellular Carcinoma and Colorectal Adenocarcinoma Cells

The formation of two nanodelivery systems, Sorafenib (SF)-loaded chitosan (SF-CS) and their folate-coated (SF-CS-FA) nanoparticles (NPs), were developed to enhance SF drug delivery on human Hepatocellular Carcinoma (HepG2) and Colorectal Adenocarcinoma (HT29) cell lines. The ionic gelation method was adopted to synthesize the NPs. The characterizations were performed by DLS, FESEM, TEM, XRD, TGA, FTIR, and UV-visible spectroscopy. It was found that 83.7 ± 2.4% and 87.9 ± 1.1% of encapsulation efficiency; 18.2 ± 1.3% and 19.9 ± 1.4% of loading content; 76.3 ± 13.7 nm and 81.6 ± 12.9 nm of hydrodynamic size; 60–80 nm and 70–100 nm of TEM; and FESEM sizes of near-spherical shape were observed, respectively, for SF-CS and SF-CS-FA nanoparticles. The SF showed excellent release from the nanoparticles under pH 4.8 PBS solution, indicating a good delivery system for tumor cells. The cytotoxicity study revealed their better anticancer action towards HepG2 and HT29 cell lines compared to the free sorafenib. Moreover, both NPs systems showed negligible toxicity to normal Human Dermal Fibroblast adult cells (HDFa). This is towards an enhanced anticancer drug delivery system with sustained-release properties for better cancer management.