Benjaporn Buranrat

Crucial role of heme oxygenase-1 on the sensitivity of cholangiocarcinoma cells to chemotherapeutic agents.

Cancer cells acquire drug resistance via various mechanisms including enhanced cellular cytoprotective and antioxidant activities. Heme oxygenase-1 (HO-1) is a key enzyme exerting potent cytoprotection, cell proliferation and drug resistance. We aimed to investigate roles of HO-1 in human cholangiocarcinoma (CCA) cells for cytoprotection against chemotherapeutic agents. KKU-100 and KKU-M214 CCA cell lines with high and low HO-1 expression levels, respectively, were used to evaluate the sensitivity to chemotherapeutic agents, gemcitabine (Gem) and doxorubicin. Inhibition of HO-1 by zinc protoporphyrin IX (ZnPP) sensitized both cell types to the cytotoxicity of chemotherapeutic agents. HO-1 gene silencing by siRNA validated the cytoprotective effect of HO-1 on CCA cells against Gem. Induction of HO-1 protein expression by stannous chloride enhanced the cytoprotection and suppression of apoptosis caused by anticancer agents. The sensitizing effect of ZnPP was associated with increased ROS formation and loss of mitochondrial transmembrane potential, while Gem alone did not show any effects. A ROS scavenger, Tempol, abolished the sensitizing effect of ZnPP on Gem. Combination of ZnPP and Gem enhanced the release of cytochrome c and increased p21 levels. The results show that HO-1 played a critical role in cytoprotection in CCA cells against chemotherapeutic agents. Targeted inhibition of HO-1 may be a strategy to overcome drug resistance in chemotherapy of bile duct cancer.

Cytoprotective effects of ferritin on doxorubicin-induced breast cancer cell death

Ferritin is a major iron storage protein and essential for iron homeostasis. It has a wide range of functions in the body including iron delivery, immunosuppression, angiogenesis, and cell proliferation. Ferritin is overexpressed in many cancer cells, but its precise role in cancer is unclear. In the present study, we examined the functional roles of ferritin in protecting the MCF-7 breast cancer cell line against treatment with the chemotherapeutic agent doxorubicin. The effects of ferritin (human liver ferritin) and doxorubicin on the human MCF-7 breast cancer cell line were evaluated using the cell viability assay. The impact of decreasing ferritin light chain (FTL) and ferritin heavy chain (FTH) expression on doxorubicin sensitivity was assessed using siRNA. Reactive oxygen species (ROS) was also measured using the fluorescence probe CM-H2DCFDA. The mechanism of modulated chemosensitivity was evaluated by western blot analysis. Ferritin treatment activated MCF-7 cell proliferation in a concentration- and time-dependent manner. While treatment with doxorubicin alone significantly increased intracelullar ROS production, the addition of ferritin decreased this ROS formation, thereby reducing doxorubicin-induced MCF-7 cell death. The inhibition of FTL and FTH by siRNA sensitized cells to doxorubicin. Treatment with doxorubicin alone significantly induced the cell cycle-dependent kinase inhibitor protein p21, whereas ferritin reduced p21 expression. Thus, ferritin plays a critical role in protecting MCF-7 cells against the chemotherapeutic drug doxorubicin. A targeted reduction of ferritin may be a useful strategy for overcoming chemoresistance in breast cancer.

Optimization of Resazurin-based Assay for Cytotoxicity Test in Cholangiocarcinoma Cells

The viability of cell cultures is usually assessed from the metabolic capability of cells in converting some chemicals to color dyes which can be conveniently measured by multi-well plate reader. However, the inconsistency between the metabolic assays and direct microscopic examination is usually observed and raised some concerns over the validity of the metabolic assay. Resazurin test, one of the metabolic assays similar to the tetrazolium MTT assay, was evaluated against a fluorescent staining microscopic counting methods. The study was performed in cholangiocarcinoma (CCA) cell line KKU-100 and cells were treated with gemcitabine, a highly potent chemotherapeutic agent. Results were revealed that cell density, types of incubation medium and incubation time of resazurin affect the consistency of the metabolic assay as compared with a microscopic counting. Optimization of resazurin-based assay is necessary to provide resazurin test as a simple, rapid and high sensitive cytotoxicity test for the anticancer drugs for cholangiocarcinoma cells. การอยรอดของเซลลเพาะเลยงมกทำการประเมนดวยการวดความสามารถในการเมแทโบลซมของเซลลโดยการเปลยนสารเคมเปนสทสามารถตรวจวดไดอยางสะดวกโดยเครองอานแพลท อยางไรกตามมกมรายงานกลาวถงความไมเทาเทยมกนของการประเมนผลระหวางวธการวดเมแทโบลซมของเซลลกบการสงเกตโดยตรงจากกลองจลทรรศน ทำใหความเปนหวงถงความถกตองของผลการอานจากการอานสโดยวธตางๆ การวเคราะหดวย resazurin ซงจดเปนการวดสจากเมแทโบลซมของเซลลเชนเดยวกบวธอนเชน tetrazolium MTT ไดนำมาศกษาเปรยบเทยบโดยวดการอยรอดของเซลลมะเรงทอนำด KKU-100ทไดรบยาเคมบำบด gemcitabine โดยการวดยอมเซลลดวยสฟลออเรสเซนทและนบเซลลภายใตกลองจลทรรศน กบวธ resazurin การศกษาพบวาจำนวนเซลล ชนดของนำยาทำปฏกรยา และเวลาของปฏกรยาของresazurinมผลตอความแมนยำของการวเคราะห เมอไดปรบปรงวธการวเคราะห ทงสองวธมประสทธภาพใกลเคยงกน ดงนน วธรซาซรนจงเปนวธทงาย รวดเรว และมความไวสง และสามารถใชเพอทดสอบความไวหรอการตอบสนองตอยาตานมะเรงของเซลลมะเรงทอนำด

Simvastatin and atorvastatin as inhibitors of proliferation and inducers of apoptosis in human cholangiocarcinoma cells.

AIMS In this study, we investigated whether statins induce human cholangiocarcinoma (CCA) cell death and apoptosis, and examined the mechanism by which statins act on cells. MAIN METHODS Four CCA cell lines, KKU-100, KKU-M055, KKU-M214, and KKU-M156 CCA cell lines were examined for HMGCR mRNA expression by the RT-PCR method. Two CCA cell lines, with low and high HMGCR mRNA expression, were used to evaluate the sensitivity to two statins, simvastatin and atorvastatin. Cytotoxic activity, antiproliferative activity, and cell migratory effects of the statins on CCA cells were evaluated using sulforhodamine B (SRB) and acridine orange/ethidium bromide (AO/EB), the colony formation assay, and wound healing assay, respectively. ROS formation was measured and apoptosis-related proteins were analyzed by Western blotting. KEY FINDINGS Both statins induced KKU-100 and KKU-M214 cell death in a time- and dose-dependent manner. Statins induced cell death more potently in the KKU-100 cells exhibiting low HMGCR expression than the KKU-M214 cells which had high HMGCR expression. Simvastatin was more potently cytotoxic than atorvastatin with lower IC50 values. Treatment with statins also caused a concentration-dependent decline in colony forming ability and cell migration. Both statins induced reactive oxygen species (ROS) formation in KKU-100 cells, but not in KKU-M214 cells. Simvastatin enhanced the release of cytochrome c, caspase 3, and increased p21 levels, especially for the KKU-100 cells. SIGNIFICANCE Statins induced CCA cell death, inhibited cell migration, and induced apoptosis. Cell death was probably induced via the mitochondrial pathway. Statins could potentially be developed as novel chemotherapeutic agents for CCA.

Simvastatin potentiates doxorubicin activity against MCF‑7 breast cancer cells

Simvastatin is a low density lipoprotein-lowering drug that is widely used to prevent and treat cardiovascular disease by inhibiting the mevalonate pathway. Simvastatin also exhibits inhibitory effects on a number of types of cancer. In the present study, the effects of simvastatin on the activity of doxorubicin in the breast cancer MCF-7 cell line, and the mechanisms by which this interaction occurs were investigated. The effect of simvastatin and doxorubicin treatment, alone and in combination, on the growth of MCF-7 cells was evaluated by a sulforhodamine B and colony formation assay. To delineate the mechanisms of cell death, the following parameters were measured: Reactive oxygen species (ROS) production using the fluorescence probe dihydroethidium; caspase 3 activity by the fluorometry method; gene expression by quantitative polymerase chain reaction; and apoptotic- and proliferative-related protein levels by western blotting. MCF-7 cell proliferation was significantly suppressed by 24-48 h treatment with simvastatin alone. Doses of 10-50 µM simvastatin also enhanced the cytotoxicity of doxorubicin against MCF-7 cells in a dose-dependent manner, and decreased the colony-forming ability of MCF-7 cells. Simvastatin alone or in combination with doxorubicin significantly increased ROS levels. Combination treatment significantly decreased expression of the cell cycle regulatory protein Ras-related C3 botulinum toxin substrate 1 and numerous downstream proteins including cyclin-dependent kinase (Cdk) 2, Cdk4 and Cdk6. Additionally, simvastatin in combination with doxorubicin significantly induced expression of the cyclin-dependent kinase inhibitor p21, increased cytochrome c and caspase 3 expression and reduced cyclin D1 expression. In conclusion, simvastatin acts synergistically with the anticancer drug doxorubicin against MCF-7 cells, possibly through a downregulation of the cell cycle or induction of apoptosis. Although additional studies are required, simvastatin and doxorubicin combination may be a reasonable regimen for the treatment of breast cancer.

Cratoxy formosum leaf extract inhibits proliferation and migration of human breast cancer MCF-7 cells

In this study we investigated how Cratoxy formosum (CF) leaf extract affects the viability and migration of human breast cancer cells including the mechanism(s) responsible. Our results showed that CF leaf extract strongly induced MCF-7 cell death in a concentration- and time-dependent manner, with IC50 values of 85.70±4.52μg/mL and 53.74±3.02μg/mL at 24h and 48h, respectively. Additionally, CF leaf extract potentiated the activity of 4 anticancer drugs with the greatest synergy occurring between CF and 5-FU. CF leaf extract also caused a dose-dependent decrease in colony forming ability with IC50 values of 36.37+1.80 μg/mL and cell migration, with IC50 values of 43.68±0.86μg/mL. Moreover, CF significantly induced ROS formation, increased caspase 3 activities, and reduced the mitochondrial membrane potential, leading to cancer cell apoptosis and cell death. In addition, the extract inhibited cancer cell migration at 25μg/mL by reducing MMP 2 and MMP 9 protein expression. Moreover, CF leaf extracts strongly decreased expression of the cell cycle regulatory protein Rac1 and downstream protein, cdk6. CF leaf extract significantly stimulated p21 and this correlated with a reduction in cyclin D1 protein levels. In summary, CF leaf extract can inhibit cell proliferation, induce cell apoptosis, and reduce cell migration in the MCF-7 cell line. It could also be beneficial for enhancing the activity of anticancer drugs used to treat breast cancer.

Investigation of antibacterial and anti-cancer activities of Streptomyces sp SRF1 culture filtrate

Purpose : To evaluate the antibacterial activity and cytotoxic effects of Streptomyces sp. SRF1 culture filtrate extract against breast cancer cell line. Methods : The activity of the extract against Gram-positive and Gram-negative bacteria was initially screened by an agar-well diffusion method. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were measured by broth microdilution method. Time-kill assays were also performed, and extract-induced morphological and ultrastructural changes to bacterial cells were investigated. Sulforhodamine B (SRB) assay was performed to determine the cytotoxicity of the extract against the human breast cancer cell line, MCF-7. Results: Antibacterial activity by the extract was detected against four strains of Gram-positive pathogens including one strain of methicillin-susceptible Staphylococcus aureus (MSSA) and 3 strains of methicillin-resistant Staphylococcus aureus (MRSA) - with low MIC and MBC values. This activity was bactericidal after 6 h exposure. Morphological alterations were detected on the cell surface of both MSSA and MRSA. The extract also inhibited MCF-7 cell growth with half-maximal concentration (IC50) of 211.67 ± 33.95 μg/mL in 72 h. Conclusions: Streptomyces sp . SRF1 culture filtrate extract exhibits potent antibacterial and anticancer activities and thus, represents a potential source of antibacterial and anticancer drugs. Keywords : Antibacterial activity, Anti-breast cancer, Staphylococcus aureus , Streptomyces sp. SRF1

Cytotoxic and antimigratory effects of Cratoxy formosum extract against HepG2 liver cancer cells

The aim of the present study was to investigate the molecular mechanisms underlying Cratoxylum formosum (CF) Dyer-induced cancer cell death and antimigratory effects in HepG2 liver cancer cells. The cytotoxic, antiproliferative and antimigratory effects of CF leaf extract on human liver cancer HepG2 cell lines were evaluated using sulforhodamine B, colony formation, and wound healing assays. In addition, apoptosis induction mechanisms were investigated via reactive oxygen species (ROS) formation, caspase 3 activities, and mitochondrial membrane potential (ΔΨm) disruption. Gene expression and apoptosis-associated protein levels were measured by reverse transcription-quantitative polymerase chain reaction and western blotting. CF induced HepG2 cell death in a time- and dose-dependent manner with half maximal inhibitory concentration values of 219.03±9.96 and 124.90±6.86 µg/ml at 24 and 48 h, respectively. Treatment with CF caused a significant and dose-dependent decrease in colony forming ability and cell migration. Furthermore, the present study demonstrated that CF induced ROS formation, increased caspase 3 activities, decreased the ΔΨm, and caused HepG2 apoptosis. CF marginally decreased the expression level of the cell cycle regulatory protein, ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1) and the downstream protein, cyclin dependent kinase 6. Additionally, CF significantly enhanced p21 levels, reduced cyclin D1 protein levels and triggered cancer cell death. CF leaf extracts induced cell death, stimulated apoptosis and inhibited migration in HepG2 cells. Thus, CF may be useful for developing an anticancer drug candidate for the treatment of liver cancer.