Co-chemotherapeutic Agent Research Articles

Cytotoxic Activity and Senescence Modulatory Effect of Hesperetin on Triple-Negative Breast Cancer Cells and Kidney Cells Co-Treatment with Cisplatin

Cisplatin (Cisp) is a non-specific chemotherapeutic agent for breast cancer. Hesperetin (HST), a flavanone found in various citrus fruits, exhibits bioactive properties, functioning as an antioxidant, anti-inflammatory, and anticancer agent. The objective of this research was to investigate the potential of HST as a co-chemotherapeutic agent in conjunction with Cisp, specifically focusing on its cytotoxic effects against 4T1 triple-negative breast cancer cells and senescence modulatory effect on Vero normal kidney cells. The cytotoxic effect and viability cell of HST were evaluated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. In addition, the effect of cellular senescence inhibition on the Vero cell line was measured using senescence-associated β-galactosidase (SA-β-gal) staining. In the MTT assay, both HST and cisplatin demonstrated a reduction in the viability of 4T1 cells in a dose-dependent manner, yielding IC50 values of 498 μM and 2 μM, respectively. The co-treatment of HST and cisplatin showed an increase in sensitivity of the 4T1 cells with a combination index of <1. HST showed low cytotoxic activity against Vero cells, with IC50 values of over 500 μM. HST decreased cellular senescence induced by cisplatin exposure on Vero cells. These results indicated that HST in co-treatment with cisplatin decreased 4T1 cell viability synergistically. HST independently reduces the cellular senescence of normal cells. Consequently, HST holds promise for potential development as a co-treatment agent in combination with cisplatin for breast cancer cells, and it may also serve as an alternative for counteracting senescence in healthy tissues.Keywords: cytotoxic, senescence, hesperetin, cisplatin, breast cancer.

The Exploration of Vetiver (Vetiveria zizanioides) as Co-Chemotherapy of Lung Cancer Selectively Targets AKR1C1: Bioinformatics Approach

Reactive Oxygen Species (ROS) is one of the cancer-causing agents, one of which is lung cancer. In addition to being carcinogenic, ROS can also be used to kill cancer cells themselves, by increasing their levels to the threshold of apoptosis. Therefore, it is necessary to inhibit certain antioxidant enzymes that are highly expressed in lung cancer. One of them is AKR1C1 which plays a role in the eradication of intracellular ROS. However, AKR1C1 has a high structural similarity to AKR1C2, so it can inhibit therapy causing selectivity problems. Vetiver (Vetiveria zizanioides) has potential as an anticancer. This study was conducted to explore vetiver as a co-chemotherapeutic agent for lung cancer targeting AKR1C1 selectively. The method used is distillation, identification of vetiver compounds using GC-MS, and through bioinformatics studies. Predictive analysis with KNIME was carried out to determine the activity of the test compound. All tested vetiver compounds had a predictive value of 1 (active) on AKR1C1 and 0 (inactive) on AKR1C2. Through GC-MS obtained 354 compounds were identified. These compounds are used to filter the compounds predicted by KNIME. The molecular docking results showed that of the 10 tested vetiver compounds, there was 1 compound that had the strongest bond in interacting with AKR1C1, namely beta vetispirene compound with an S-score of -15.12 kcal/mol, and stronger than native ligand and aspirin. Based on the research data, it can be concluded that the beta vetispirene compound in vetiver can be a potential co-chemotherapy agent for lung cancer in targeting AKR1C1 selectively. However, further research is needed to prove its activity on lung cancer cells.Keywords: ROS (Reactive Oxygen Species), Lung Cancer, AKR1C1, selectivity, vetiver (Vetiveria zizanioides).

Parkia speciosa Seeds Ethanol Extract as Co-chemotherapeutic Agent for Doxorubicin Toward Tongue Cancer

BACKGROUND: Parkia speciosa seeds have been reported to have an anticancer property due to the presence of various antioxidant compounds. Since the potential uses of P. speciosa for the tongue cancer has not been clearly disclosed, we conducted a study to investigate anticancer properties of P. speciosa seed ethanol extract (PSSEE) as well as its effect on cardiac cells.METHODS: Tongue cancer rat model were treated with/without Doxorubicin and various concentrations of PSSEE. After treatment, tongue and heart samples were collected and processed further for histological examinations. Tongue epithelium thickness and damaged heart tissues was observed by HE staining, while tongue cancer cell proliferation was assessed by Ki-67 immunohistochemistry. Analyses were performed under an upright light microscope to measure tongue epithelium thickness, state of cancer cell proliferation, and degree of heart tissue damage.RESULTS: Addition of 400 mg/kg body weight (BW) PSSEE to 4.6 mg/kg BW Doxorubicin reduced the average tongue epithelial thickness and Ki-67+ cells number. Upon addition of PSSEE to Doxorubicin, the damage of heart tissue was reduced in a concentration dependent manner. Among all groups, the group of tongue cancer treated with 4.6 mg/kg BW Doxorubicin and 400 mg/kg BW PSSEE had the lowest percentage as well as the lowest degree of heart tissue damage.CONCLUSION: Since addition of PSSEE to Doxorubicin reduced epithelial thickness, number of Ki-67+ cells and heart tissue damage, PSSEE could be a potential co-chemotherapeutic agent for Doxorubicin toward tongue cancer.KEYWORDS: Parkia speciosa, Doxorubicin, tongue cancer, epithelial thickness, Ki-67, cardiotoxicity, co-chemotherapy

Solanum nigrum Ethanolic Extract (SNE) Increases Cytotoxic Activity of Doxorubicin on MCF-7 Cell

Leunca (Solanum nigrum L.) is a potential source of natural anticancer agents. Solanum nigrum L. ethanolic extract (SNE) has cytotoxic activity in several cancer cell lines. We aimed to evaluate the ability of SNE to increase MCF-7 cell sensitivity to doxorubicin as a chemotherapeutic agent for breast cancer. Cell viability of SNE and its combination treatment with doxorubicin were conducted by 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) assay, and apoptosis assay was analyzed by Ethidium bromide-acridine orange method. The SNE showed a cytotoxic effect in the MCF-7 cell line with IC50 50 μg/mL. Combination treated DOX-SNE resulted in a combination index (CI) value of 0.21, indicating strong synergism SNE and doxorubicin. The SNE 25 μg/mL combined with doxorubicin 100 nM optimally induced apoptosis of MCF-7 cells. We concluded that SNE is the potential to be developed as a co-chemotherapeutic agent through apoptosis induction though the molecular mechanism need to explore.Keywords: Solanum nigrum L. herb ethanolic extract, doxorubicin, MCF-7, apoptosis.

Citrus sinensis (L) Peels Extract Inhibits Metastasis of Breast Cancer Cells by Targeting the Downregulation Matrix Metalloproteinases-9

Introduction:
 Long-term use of doxorubicin (DOX) chemotherapy causes several side effects, especially induction of metastasis on breast cancer (BC). There is an urgent need to identify novel agent with low side effect targeting BC metastasis. Citrus sinensis (L.) peels extract (CSP) has long been used for the treatment of several cancer. However, its anti-metastatic potential against BC metastatic remains unclear.
 Objective:
 This study aimed to explore the role of CSP in combination with DOX in inhibiting the migration of metastatic breast cancer MDAMB-231 cells.
 Material and Methods:
 Potential cytotoxic in single and combination was analysed 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay). The anti-metastatic activities of several major compound on CSP including hesperetin, tangeretin, nobiletin, naringenin and hesperidin were screened by molecular docking under PLANTS software.
 Results:
 Based on molecular docking we revealed that the selected protein target MMP-9 (PBD ID:2OVX) has lower docking score for hesperetin, tangeretin, nobiletin, naringenin and hesperidin compare to DOX. CSP and DOX individually exhibited strong cytotoxic effect on MDA-MB-231 cells under MTT assay with IC50 value of 344 µg/mL and 85 nM, respectively. Furthermore, CSP in combination with DOX synergistically increased the cytotoxicity of DOX. Here, we showed that CSP can specifically suppress the side effect of DOX-induced metastasis by reduces doses of DOX. However, low doses of DOX in combination with CSP still potential inhibited cancer cells growth.
 Conclusion:
 In conclusion, CSP increased the cytotoxicity and inhibited the induction of metastasis by DOX in breast cancer cells. So that, CSP potential to be developed as co-chemotherapeutic agent.

Cinnamon oil as a co-chemotherapy agent through inhibition of cell migration and MMP-9 expression on 4T1 cells.

The long-term and high-dose use of doxorubicin as chemotherapy for triple-negative breast cancer (TNBC) patients induces epithelial-to-mesenchymal transition (EMT) and stimulates cancer metastasis. Cinnamaldehyde is a major compound of cinnamon oil (CO) suppressing Snail and NFκB activity that are involved in cell migration. This study aims to explore the activity of CO as a co-chemotherapeutic agent on 4T1 breast cancer cells. The CO was obtained by water and steam distillation and was characterized phytochemically by gas chromatography-mass spectrometry (GC-MS). Cytotoxic activity of single CO or in combination with doxorubicin was observed by MTT assay. Cell migration and MMP-9 expression were measured by scratch wound healing and gelatin zymography assays. The intracellular reactive oxygen species (ROS) levels were observed by 2',7'-dichlorofluorescin diacetate (DCFDA) staining flowcytometry. The phytochemical analysis with GC-MS showed that CO contains 14 compounds with cinnamaldehyde as the major compound. CO exhibited cytotoxicity on 4T1 cells with the IC50 value of 25μg/mL and its combination with doxorubicin decreased cell viability and inhibited cell migration compared to a single use. Furthermore, the combination of CO and doxorubicin inhibited MMP-9 expression and elevated intracellular ROS levels compared to control. CO has the potential to be developed as a co-chemotherapy agent through inhibition of cell migration, and intracellular ROS levels elevation.

Reactive oxygen species and senescence modulatory effects of rice bran extract on 4T1 and NIH-3T3 cells co-treatment with doxorubicin

Objective: To determine the effect of rice bran extract (RBE) in combination with doxorubicin on 4T1 triple-negative breast cancer cells and NIH-3T3 cells. Methods: RBE was obtained by maceration with n-hexane. The phytochemical profile of RBE was observed using highperformance liquid chromatography. Cytotoxic effect of RBE was evaluated through MTT assay. In addition, flow cytometry was used for cell cycle and apoptosis analysis. Cellular senescence was observed using SA-β-Gal assay and intracellular reactive oxygen species (ROS) levels were evaluated using DCFDA staining. The pro-oxidant property of RBE was also evaluated through 1-chloro- 2,4-dinitrobenzene spectrophotometry and molecular docking. Results: RBE was obtained with a yield of 18.42% w/w and contained tocotrienols as the major compound. RBE exerted no cytotoxic effect on 4T1 and NIH-3T3 cells. However, RBE in combination with doxorubicin decreased 4T1 cell viability synergistically (combination index<0.9) and induced apoptosis and senescence on 4T1 cells. RBE significantly decreased senescence in doxorubicin-treated NIH-3T3 cells. Additionally, RBE did not increase ROS levels in doxorubicin-treated 4T1 cells. Meanwhile, the combination of RBE and doxorubicin reduced ROS levels in NIH-3T3 cells. RBE significantly reduced glutathione-S-transferase activity and alpha-tocotrienol interacted with glutathione-S- transferase in the glutathione binding site. Conclusions: Rice bran may be used as a co-chemotherapeutic agent to improve the therapeutic effectiveness of doxorubicin while protecting against the cellular senescence effects of doxorubicin on healthy cells.

Co-treatment of Brazilein Enhances Cytotoxicity of Doxorubicin on WiDr Colorectal Cancer Cells Through Cell Cycle Arrest

BACKGROUND: The presence of adverse side effects limits the use of doxorubicin (Dox) despite its cost-effectiveness compared to other chemotherapeutic agents. Brazilein (Be), the major compound of Caesalpinia sappan, performs co-chemotherapeutic potency in several cancer cell lines. This study evaluates the chemosensitizing effects of Be to Dox on colon cancer cell line, WiDr.METHODS: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was conducted to evaluate the cytotoxic effect of Be and its combination with Dox. The synergistic effect of Be and Dox was examined by using the Combination index (CI) parameter. Cell cycle and apoptosis profiles were done using flow cytometry with propidium iodide (PI)/RNase and Annexin V staining, respectively.RESULTS: The combination of Dox and Be at half of IC50 on WiDr cells showed a synergistic effect with a combination index of 0.4. Analysis of the cell cycle revealed that the combination caused cell cycle termination at the S and G2/M phase. This finding corresponded with the data that single treatment of Dox and Be induced cell cycle arrest at the different phases, namely S and G2/M phase, respectively. However, the combination treatment for 24 hours did not induce apoptosis. This combination should be further clarified as there was a possibility that many cells may underwent permanently arrest that halts to proceed apoptosis.CONCLUSION: Our findings suggested that Be synergizes with Dox to suppress the growth of WiDr cells via cell cycle arrest, hence, Be is potential to be developed as a co-chemotherapeutic agent. Our findings suggested that Be synergizes with Dox to suppress the growth of WiDr cells via cell cycle arrest, hence, Be is potential to be developed as a co-chemotherapeutic agent.KEYWORDS: Brazilein, colon cancer WiDr, co-treatment, Doxorubicin, cell cycle arrest

Cytotoxic and Antimetastatic Activity of Hesperetin and Doxorubicin Combination Toward Her2 Expressing Breast Cancer Cells

Objective:This study aimed to explore Hesperetin (Hst) potency as a co-chemotherapeutics agent combined with Doxorubicin (Dox), particularly cytotoxic and antimetastasis effects toward MCF-7/HER2 cells. Methods:The cytotoxic effects were measured under MTT assay. The flowcytometry analysis was used to examine the cell cycle modulation and apoptosis evidence, while the effect of migration was assayed by scratch wound healing assay. Western blotting and gelatin zymography were carried out to examine the expression level of proteins, HER2, and Rac1. Results:Under MTT assay, Hst and Dox exhibited to decrease cell viability in a dose-dependent manner with the IC50 value of 377 and 0,8 µM, respectively. The combination of Hst and Dox at the respective doses of 95 and 0,2 µM showed a synergistic effect with the combination index of 0,63. Flow cytometry analysis of Hst-Dox revealed that those compounds caused cell cycle arrest at the G2/M phase and induced apoptosis. Hst also decreased HER2 and Rac1 expression, as shown by western blot. Hst inhibited lamellipodia formation and cell migration, as indicated by microscopic observation and wound healing scratch assay. The antimetastatic activity of Hst was associated with the reduction of Rac1 and MMP9 expression as measured by gelatine zymography assay. Conclusion:These results indicated that the combination of Hst and Dox-induced cell cycle arrest, apoptosis, decreased HER2, Rac1, MMP9 expression, and cell migration. Thus, Hst may have the potential to be developed as a co-chemotherapeutic agent combined with doxorubicin toward HER2 overexpressing breast cancer cells.

Effects of Peel Extract from Citrus reticulata and Hesperidin, A Citrus Flavonoid, on Macrophage Cell Line

The extract of Citrus reticulata has been studied for its biological activities, due to its citrus flavonoid content. The extract and its flavonoid compounds exhibit growth inhibition property in several cancer cell lines and in vivo models. Conversely, the extract can also induce cell proliferation and angiogenesis, and shows estrogenic effects by in vitro and in vivo. Because of the contrasting effects that depend on the concentration or dosage, the precise action of the extract and its flavonoids need to be elucidated in various cell types. The objective of this study is to evaluate the effect of Citrus reticulata peel extract (Citrus extract) and hesperidin, a citrus flavonoid, on the modulation of cell proliferation in the RAW 264.7 macrophage cell line. Cell viability under Citrus extract or hesperidin treatment was assessed by using the MTT assay. The expression of interleukin-10 (IL-10), an anti-inflammatory cytokine, modulated by Citrus extract was also examined by immunostaining. Low concentrations of Citrus extract at 1 and 100μg/mL were able to induce cell proliferation, although in significant, as shown by cell viability of 138 and 114%, respectively. At higher concentrations of 500, 750, and 1000μg/mL, Citrus extract decreased cell viability significantly by up to 64, 46, and 36%, respectively. Accordingly, hesperidin at low (3.1μg/mL −61.1μg/mL) concentration increased cell viability significantly by up to 116-136% where as high (152.6203μg/mL–305.3203μg/mL) concentration reduced cell viability significantly by up to 10-61%. The value of the 50% inhibitory concentration (IC50) of Citrus extract was more than three times higher (756μg/mL) than that of hesperidin (203μg/mL = 332μM). Additionally, 250μg/mL of Citrus extract was able to induce IL-10 expression compared to control. These results demonstrated that Citrus extract and hesperidin exerted a biphasic effect on macrophage cells. The future development of Citrus extract as a co-chemotherapeutic, anticancer, or immunomodulatory agent should include careful consideration of its biphasic effect on each cell type

Revealing the Reversal Effect of Galangal (Alpinia galanga L.) Extract Against Oxidative Stress in Metastatic Breast Cancer Cells and Normal Fibroblast Cells Intended as a Co- Chemotherapeutic and Anti-Ageing Agent.

Objective:This study intends to explore the potential of galangal extract as a co-chemotherapeutic agent through the analysis of its cytotoxic and migratory effects on metastatic breast cancer cells and as an anti-ageing agent through its senescence inhibitory effect on normal fibroblast cells. Methods:Galangal ethanolic extract (GE) was subjected to a cytotoxicity test with the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay alone or in combination with doxorubicin (Dox) against 4T1 cells but not in NIH-3T3 cells. Evidence of senescent cells was detected using a SA-β galactosidase based assay. In addition, the level of reactive oxygen species (ROS), apoptosis, and cell cycle were measured with a flow cytometry-based assay. Meanwhile, cell migration and matrix metalloproteinase (MMP)-9 expression after GE treatment on 4T1 cells were measured using the scratch wound healing assay and gelatin zymography assay, respectively. The metabolomic profiles of GE were traced using gas chromatography-mass spectrometry (GC-MS) analysis. Results:GE effectively inhibited the growth of 4T1 cells with an IC50 value of 135 µg/mL and increased the cytotoxic effect of Dox at concentrations of 50 and 100 µg/mL. GE increased the number of senescent cells arrested in the G2/M phase but did not cause apoptosis. This effect is compounded by increasing intracellular levels of ROS. However, GE reduced senescence to normal in fibroblast cells (NIH 3T3 cells) under oxidative stress by Dox without any changes in the ROS level. Moreover, GE also inhibited the migration of 4T1 cells and suppressed the expression of MMP-9 induced by Dox. Conclusion:Galangal has the potential for use as a co-chemotherapeutic agent by inducing senescence in correlation with increasing intracellular ROS toward metastatic breast cancer. However, the effect of GE in decreasing the senescence phenomena toward normal fibroblast cells illustrates its potential as a promising anti-ageing agent.

COMBINATION OF HEDYOTIS CORYMBOSA L. AND TINOSPORA CRISPA ETHANOLIC EXTRACT INCREASE CISPLATIN CYTOTOXICITY ON T47D BREAST CANCER CELLS

Objective: Ursolic acid was a compound found in Hedyotis corymbosa L., (HCoL) while berberine found in Tinospora crispa (TCa) which are proven to have cytotoxic effect to cancer cells. This research aims to review the effect of cisplatin, ethanolic extract of HCoL and TCa to the sensitivity increase on breast cancer cells, which will be confirmed through apoptosis induction and cell cycle modulation.Methods: The cytotoxic effect was tested using 3-(4,5-dimethylthiazol-2-il)-2,5-diphenyltetrazolium bromide assay on T47D cell using the IC50 parameter. The combination was tested by determining their combination index (CI) and cell viability. The combination effect of apoptosis induction and cell cycle modulation was observed using flow cytometry method.Results: The cytotoxic test result of the combination shows CI value of below 1 at the concentration of HCoL ethanolic extract as much as 1 μg/mL, TCa ethanolic extract as much as 6 μg/mL, and cisplatin as much as 2,5 μM. The combination of HCoL ethanolic extract, TCa ethanolic extract, and cisplatin results in phase S cell accumulation (29.98%) on breast cancer cell T47D and was able to induce apoptosis.Conclusion: The result proves that ethanolic extract of HCoL and TCa can be developed as a cochemotherapeutic agent with cisplatin to increase the effectivity of breast cancer treatment.

EFFECT OF HEXANE FRACTION FROM PAPAYA (CARICA PAPAYA L.) MALE FLOWER ON CELL CYCLE OF COLON ADENOCARCINOMA (WIDR) CELL AND ITS COMBINATION INDEX WITH DOXORUBICIN

Objective: This study aimed to evaluate the effects of papaya (Carica papaya L.) male flower hexane fraction (PHF) on cell cycle of colon adenocarcinoma (WiDr) cell and its combination index (CI) with doxorubicin.Methods: Flow cytometer and the CI were used to show the PHF on cell cycle of colon adenocarcinoma (WiDr) cell and to calculate the synergism potential, respectively.Result: The result showed that the PHF giving inhibition on cell cycle of colon adenocarcinoma (WiDr) cell in G0-G1, S, G2-M phase. Furthermore, the combination of PHF with doxorubicin in colon adenocarcinoma (WiDr) cell gave a strong synergistic effect with optimal concentration of 8 μg/ml–50 nM (PHF-Doxorubicin) with the IC score lower than 1.Conclusion: This study provides evidence that PHF could be a new potential co-chemotherapeutic agent with doxorubicin on colon adenocarcinoma cell.

The aqueous extract of Gerrardanthus macrorhizus caudex enhanced doxorubicin activity in MCF-7 human breast cancer cells

Gerrardanthus macrorhizus (GM) caudex, is traditionally used in cancer therapy by the Tetun people in Belu District, East Nusa Tenggara Province, Indonesia, where it is known as “akar batu”. This study aimed to explore the cytotoxic effects of G. macrorhizus caudex aqueous extract, as well as its combination with doxorubicin, on MCF-7 cells. Also investigated were the possible mechanisms of interaction through cell cycle progression and apoptosis induction. Single treatments of 5–320 µg/mL of the extract showed morphological alterations in MCF-7 cells, but did not show any cytotoxic effect. Combining the extract with doxorubicin resulted in a synergistic cytotoxic effect. Doxorubicin concentrations equivalent to 1/12, 1/8, and 1/5 fold of the IC50 combined with 20 µg/mL decreased viability to 48%. We then explored the combination effect of doxorubicin 0.4 µM with GM 5 and 20 µg/mL using a flow cytometer. A low concentration of the extract (5 µg/mL) combined with 0.4 µM of doxorubicin resulted in slight cell cycle modulation by G1, G2M arrested and apoptosis induction. The combination of doxorubicin and a higher concentration of the extract (20 µg/mL) did not show cell cycle modulation, and led to necrosis. Therefore, G. macrorhizus caudex at low concentrations has the potential to be developed further as a co-chemotherapeutic agent.

Hedyotis corymbosa L. and Sterculia quadrifida R.Br Ethanolic Extract Enhances Cisplatin’s Cytotoxicity on T47D Breast Cancer Cells Through Cell Cycle Modulation

Chemotherapeutic agent such as cisplatin used as cancer cure with side effects and resistance. Ursolic acid is a compound found in Hedyotis corymbosa L., (HCoL) while 8-hydroxydehydrodunnione is found in Sterculia quadrifida R.Br (SQuR) which are proven to have cytotoxic effect to cancer cells. This research aims to review the effect of cisplatin, ethanolic extract of HCoL and SQuR to sensitivity increase on breast cancer cells, which will be confirmed through apoptosis induction and cell cycle modulation. The cytotoxic effect was tested using MTT assay on T47D cell by using IC 50 parameter. The combination was tested by determining their combination index (CI) and cell viability. The combination effect in apoptosis induction and cell cycle modulation were observed using flow cytometry method. The cytotoxic test result of the combination shows CI value of below 1 at the concentration of HCoL ethanolic extract as much as 0,21 µg/mL, SQuR ethanolic extract as much as 0,45 µg/mL, and cisplatin as much as 2,5 µM. The combination of HCoL ethanolic extract, SQuR ethanolic extract, and cisplatin results in phase S cell accumulation (27,73%) on breast cancer cell T47D and was able to induct apoptosis. The result proves that ethanolic extract of HCoL and SQuR can developed as a co-chemotherapeutic agent to increase the effectivity of breast cancer treatment.

Red Betel Leaves Methanolic Extract (Piper crocatum Ruiz & Pav.) Increases Cytotoxic Effect of Doxorubicin on WiDr Colon Cancer Cells through Apoptosis Induction

Doxorubicin is a chemotherapeutic agent that causes a lot of side effects in high doses. Thus, combination with a co-chemotherapeutic agent which can increase its toxicity on cancer cells, is needed to reduce its therapeutic dose. Red betel leaves (Piper crocatum Ruiz & Pav.) have been known to contain flavonoids and alkaloids that have anticancer activity. This study was conducted to determine the cytotoxic effect and apoptosis induction of red betel leaves methanolic extract (RBM), doxorubicin (dox) and the combination of them on WiDr cells as model of colon cancer. RBM was extracted by soxhlet method using methanol. Cytotoxicity assay was performed using MTT assay for both single and combination treatments for 24 hours to determine IC50 and CI as their parameters. Apoptosis induction was analyzed by double staining method using ethidium bromide-acridine orange staining. Treatment of RBM and dox on WiDr cells for 24 hours showed cytotoxic activity with IC50 100 μg/mL and 1.6 μM respectively. Combination of RBM and dox performed synergism effect with CI<0.9 (p<0.05). Combination of RBM (12.5 μg/mL) and dox (0.4 μM) increased the number of apoptosis cells compared to each single treatment. Based on this study, it can be concluded that red betel leaves methanolic extract is potential to be developed as a co-chemotherapeutic agent of doxorubicin on colon cancer but still need further study to disclose the underlying molecular mechanisms.Keywords : Red betel leaves (Piper crocatum Ruiz & Pav.), doxorubicin, WiDr cells, co- chemotherapeutic agent

Pentagamavunone-0 (PGV-0), a Curcumin Analog, Enhances Cytotoxicity of 5-Fluorouracil and Modulates Cell Cycle in WiDr Colon Cancer Cells

The use of 5-fluorouracil (5-FU) in colon cancer as the primary chemotherapy has not been meet satisfactory effectiveness. Therefore, the development of new chemicals as a chemopreventive agent and a combination agent (co-chemotherapeutic agent) for colon cancer is important. Pentagamavunone-0 (2,5-bis-(4'-hydroxy-3'-methoxybenzylidine) cyclopentanone) (PGV-0), one of curcumin analogs, exhibits cytotoxic effect and apoptosis induction in various cancer cell lines, including colon cancer cell, better than curcumin. This study aimed to investigate the cytotoxic potency of PGV-0 in combination with 5-FU and their effects, in single or in combination, on cell cycle toward WiDr colon cancer cell line. The cells were treated with combination concentrations of PGV-0 and 5-FU, and examined by MTT cell viability assay. The value of combination index (CI) as a parameter of cytotoxic combination assay was measured by a combination index method. Cells were stained with propidium iodide and the cell cycle distribution was determined by flowcytometry. CI calculation showed additive effects between PGV-0 and 5-FU. Combination of PGV-0 and 5-FU gave synergism on cell cycle. Single treatment of PGV-0 increased apoptosis, illustrated as subG1-phase accumulation, stronger than single treatment of 5-FU. Meanwhile, combination of PGV-0 and 5-FU demonstrated S-phase arrest. Based on these results, it can be concluded that PGV-0 has the potential to be developed as a co-chemotherapeutic agent for colon cancer but still requires further tracking of its molecular mechanisms.Keywords: Pentagamavunone-0 (PGV-0), 5-fluorouracil (5-FU), colon cancer,combination, cell cycle

The Synergistic Effect of Doxorubicin and Ethanolic Extracts of Caesalpinia sappan L. Wood and Ficus septica Burm. f. Leaves on Viability, Cell Cycle Progression, and Apoptosis Induction of MCF­7 Cells

Caesalpinia sappan L. and Ficus septica Burm. f known asa potential plant with wide variety of medicinal properties, including anticancer. Present study was aimed to explore cytotoxic effect ofsappan wood (ECS) and awar-awar leaves (EFS), and its combination with doxorubicin (dox) on MCF-7 cells focusing on cell cycle progression and apoptosis induction.The result of MTT assay showed that single treatment of ECS and dox performed cytotoxic effect with the IC50 value of 32 µg/mL and 6 µM respectively, while EFS performed low cytotoxic effect with the IC50 value of 282 µg/mL. The combination of ECS with EFS and doxorubicin showed synergistic cytotoxic effect. Flow cytometry analysis revealed that combination of ECS (16 µg/mL) with EFS (8 µg/mL) and doxorubicin (2 µM) induced apoptosis, and cell accumulation at sub-G1 and G2/M phases.Immunoblotting assay confirmed the apoptosis induction of this combination through increasing of cleavage of PARP-1. Based on these results, the synergistic cytotoxic effect of this combinationwas through G2/M phase accumulation and apoptosis inductionand potentially to be developed as co-chemotherapeutic agent.

Synergistic Effect of Arecoline in Combination with Doxorubicin on HeLa Cervical Cancer Cells

Arecoline, the main alcaloid of Areca cathecu L has been proven to posses cytotoxic activity against various cancer cell lines. The research conducted to examine the cytotoxic activity of arecoline alone and its combination with doxorubicin against HeLa cervical cancer cell line. Single treatment of arecoline in various concentration on HeLa cancer cell were done followed by the combinational treatment with doxorubicin. The cell viability as the parameter of cytotoxicity was measured using MTT (3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolium bromide) assay. The apoptotic effect was examined by double staining assay using etidium bromide–acridin orange. Arecoline did not show potent cytotoxicity effect against HeLa since the value of IC50 is 462 µM. The combinational treatment of arecoline and doxorubicin showed synergicity with the optimum CI value is 0,48 given by the treatment of 30mM arecoline combined with 125 nM doxorubicin. The result of this study shows that arecoline has potential to be proposed as co-chemotherapeutic agent for cervical cancer. However, further study on its molecular mechanism needs to be conducted. Keywords: cinnamon essential oil, doxorubicin, T47D cells, combination cytotoxicity

PGV-0 AND PGV-1 INCREASED APOPTOSIS INDUCTION OF DOXORUBICIN ON MCF-7 BREAST CANCER CELLS

As chemotherapeutic backbone for breast cancer therapy, doxorubicin showed various side effects and induced resistancy of breast cancer cells. Development of targeted therapy on breast cancer focused on combinatorial therapy of doxorubicin and molecular targeted agents. PGV-0 and PGV-1, a curcumin analogue showed potency as co-chemotherapeutic agent with doxorubicin. Our previous study of PGV-0 and PGV-1 showed cytotoxic activity in T47D cells. Therefore, the aim of this research is to examine the synergistic effect of PGV-0, PGV-1 on the cytotoxic activity of doxorubicin through cell cycle modulation and apoptotic induction on MCF-7 breast cancer cell lines. The cytotoxic assay of PGV-0, PGV-1, doxorubicin, and their combination were carried out by using MTT assay. Cell cycle distribution and apoptosis were determined by flowcytometer FACS-Calibur and the flowcytometry data was analyzed using Cell Quest program. Single treatment of PGV-0, PGV-1 and doxorubicin showed cytotoxic effect on MCF-7 with cell viability IC50 value 50 µM, 6 µM and 350 nM respectively. Single treatment of Doxorubicin 175 nM induced G2/M arrest. Single treatment of PGV-0 5 µM induced G2/M arrest while in higher dose 12.5 µM, PGV-0 induced apoptosis. Combination of doxorubicin 175 nM and PGV-0 5 µM induced apoptosis. Combination of doxorubicin 175 nM and PGV-0 12.5 µM also increased apoptosis induction. Single treatment of PGV-1 0.6 µM induced G1 arrest while in higher dose 1.5 µM, PGV-1 induced apoptosis. Combination of doxorubicin 175 nM and PGV-1 0.6 µM induced apoptosis. Combination of doxorubicin 175 nM and PGV-0 1.5 µM also increased apoptosis induction. PGV-0 and PGV-1 are potential to be delevoped as co-chemotherapeutic agent for breast cancer by inducing apoptosis and cell cycle modulation, but the molecular mechanism need to be explored detail. Key words: PGV-0, PGV-1, doxorubicin, co-chemotherapy, breast cancer, cell cycle arrest, apoptosis