Identifikasi Isolat Ekstrak Etanol Kulit Batang Rhizophora apiculata Blume dan Rhizophora mucronata Lam Serta Sitotoksisitasnya Terhadap Sel MCF-7 dan T47D

Mangrove plants as traditional medicine have long been used by the society for the therapy of anticancer diseases. So far, the potency of mangrove plants as anticancer has not been studied intensively. The methanol extract of R. mucronata stem bark has cytotoxic activity on myeloma cells. This study aims to determine the isolates of the ethyl acetate fraction of the methanol extract of the stem bark of R. mucronata and R. apiculata, and to examine the potency of cytotoxic activity against MCF-7 and T47D cells. The powder of the stem bark of R. mucronata and R. apiculata were extracted with methanol by maceration, and then the methanol extract was fractionated consecutively using n-hexane, ethyl acetate, and methanol. Ethyl acetate fraction was then purified by column chromatography with a diameter of 2 cm and a height of 30 cm with a static phase in the form of silica gel 60 (0.063-0.2 mm) Merck 7734 and eluent n-hexane:ethyl acetate (6: 4, 5: 5, 6: 4, 7: 3, 8: 2, 9: 1) and n-hexane: methanol (9,5: 0,5) 10 ml each. The eluates that produce the same number and pattern of stains were combined into one isolate. Then the eluate was evaporated at room temperature. Then the purification results of the extract R. mucronata were isolated using preparative TLC with 0.25 mm GF254 silica gel as static phase and n-hexane mobile phase: ethyl acetate (6: 4), while the apiculata extract with n-hexane mobile phase: ethyl acetate (5: 5). The results of the stain separation of the compound were scraped off and separated from the static phase using a solvent. The purity of isolates compound was examined using TLC in the static phase of silica gel GF254 0.25 mm. The qualitative test results of the ethyl acetate fraction of R. apiculata stem bark with an NMR spectrophotometer showed a composition of cis-3- (3,5-dihydroxyphenyl) acrylic acid, while the ethyl acetate fraction of R. mucronata stem bark contained n-hexan-3-ol compounds. The cytotoxicity test of ethanol extract, n-hexane fraction, ethyl acetate fraction and ethanol extract was carried out on MCF-7 and T47D cells using the MTT method. The results proved that the ethanol extract, n-hexane fraction, ethyl acetate fraction and ethanol fraction from the ethanol extract of R. mucronata and R. apiculata stem bark, based on cytotoxic activity, were inactive against MCF-7 and T47D cells.

Cytotoxic Effect of Capsicum annum L. extract on T47D Cells: In vitro Study

Capsicum annum L. is a potential natural plant that have a lot of various pharmacological effects, including as anticancer agent. This study Aim to analyze Capsicum annum extract (CAE) on T47D cells. CAE (10, 20, 40, 60, 80µg/mL) treated on T47D cells to determined IC50 value by MTT assay. Apoptosis induction is also investigated through caspase-3 expressions (IC50, 2IC50). The present study showed that CAE suppress T47D cells proliferation with IC50 value of 75.81µg/mL. The caspase-3 expression on 2IC50 is higher (67.16%) than IC50 (52.16%). This result indicate that CAE has ability as anticancer agent by inhibiting cell growth and induce apoptosis through caspase-3 expression on T47D cells. Further study of CAE holds potential for novel therapies of cancer prevention and treatment.

MPEG-PCL Nanomicelles Platform for Synergistic Metformin and Chrysin Delivery to Breast Cancer in Mice

Background: Metformin (MET) is a well-known anti-diabetic drug that also has anti-cancer effects. However, high therapeutic doses of MET on cancer cells and the low efficacy of combinatory therapeutic approaches limit its clinical application. Recent studies have shown that chrysin (CHR) can improve the pharmaceutical efficacy of MET by suppressing human telomerase reverse transcriptase (hTERT) and cyclin D1 gene expression. Objective: This study aimed to develop different ratios of methoxy poly(ethylene glycol)-b-poly(e-caprolactone) (MPEG-PCL) micelles for breast cancer to co-deliver a synergistic CHR/MET combination. Methods: CHR/MET drug-loaded micelles were prepared by modified thin-film hydration. Fourier infrared spectrum, gel permeation chromatography, transmission electron microscopy, and high-performance liquid chromatography were used to evaluate the physicochemical properties of nanostructures. Cell proliferation and cell apoptosis were assessed by MTT and Annexin V-FITC/PI double staining method. The gene expression of hTERT and cyclin D1 was measured by real-time PCR assay. A subcutaneous mouse T47D xenograft model was established to evaluate the in vivo efficiency. Results: When the ratio of MPEG-PCL was 1:1.7, the highest drug loading rate and encapsulation efficiency of CHR (11.31±0.37) and MET (12.22±0.44) were observed. Uniform MPEG-PCL micelles of 51.70±1.91 nm allowed MET to incorporate with CHR, which were co-delivered to breast cancer cells. We demonstrated that CHR/MET co-delivery micelles showed a good synergistic effect on inhibiting proliferation in T47D cells (combination index=0.87) by suppressing hTERT and cyclin D1 gene expression. Compared with the free CHR/MET group, the apoptosis rate on T47D cells by CHR/MET nano-micelles significantly improved from 71.33% to 79.25%. The tumour volume and tumour weight of the CHR/MET group increased more slowly than that of the single-drug treatment group (P<0.05). Compared with the CHR/MET group, the tumour volume and tumour weight of the CHR/MET nano-micelle group decreased by 42% and 59%, respectively. Conclusions: We demonstrated that ratiometric CHR/MET micelles could provide an effective technique for the treatment of breast cancer.

3,3’-Diindolylmethane Enhances Paclitaxel Sensitivity by Suppressing DNMT1-Mediated KLF4 Methylation in Breast Cancer

Paclitaxel (PTX) is a first-line chemotherapeutic drug for the treatment of breast cancer, but drug resistance seriously limits its clinical use. The aim of the present work was to explore the effect of 3,3’-diindolylmethane (DIM) on PTX sensitivity and its possible mechanism in breast cancer. The expression of Krüppel-like factor 4 (KLF4) and DNA-methyltransferase 1 (DNMT1) in breast cancer tissues were assessed by immunohistochemistry and Western blotting. The methylation of KLF4 was evaluated by the MassARRAY platform. The lentivirus carrying KLF4 and DNMT1 gene or shRNA targeting DNMT1 were used to overexpress KLF4 or knockdown DNMT1 in MCF-7 and T47D breast cancer cells and the role of KLF4 and DNMT1 in regulation of PTX sensitivity was investigated. The effect of PTX on inhibiting the proliferation of MCF-7 and T47D cells was measured by CCK-8 assay. Flow cytometry was used to examine cell apoptosis. The expression of mRNA and protein was evaluated by qRT-PCR and Western blotting analysis, respectively. Our data showed that the expression of DNMT1 was increased, and the methylation level of CpG sites (−148 bp) in the KLF4 promoter was increased while the KLF4 expression was significantly decreased in breast cancer tissues. Overexpression of KLF4 increased the sensitivity of MCF-7 and T47D cells to PTX. DNMT1 increased the methylation of the KLF4 promoter and decrease the expression of KLF4. Knockdown of DNMT1 increased the sensitivity of MCF-7 and T47D cells to PTX. DIM enhanced the PTX sensitivity of MCF-7 and T47D cells, decreased the expression of DNMT1 and the methylation level of KLF4 promoter, thus increasing the level of KLF4. Furthermore, overexpression of DNMT1 attenuated the effect of DIM on the regulation of PTX sensitivity. Collectively, our data indicated that DNMT1-mediated hypermethylation of KLF4 promoter leads to downregulation of KLF4 in breast cancer. The level of KLF4 is correlated with the sensitivity of MCF-7 and T47D cells to PTX. DIM could enhance the antitumor efficacy of PTX on MCF-7 and T47D cells by regulating DNMT1 and KLF4.

A bioactive compound isolated from Duku (Lansium domesticum Corr) fruit peels exhibits cytotoxicity against T47D cell line

Background: Breast cancer is a major health problem for women globally. Many attempts have been promoted to cure cancer by finding new anticancer medicines from natural resources. Despite the richness of biodiversity discovered, there are some natural resources that remain unexplored. Fruit peels of Duku (Lansium domesticum Corr.) are rich with compounds that may have the potential to be developed as anticancer drugs. This study aimed to isolate cytotoxic compounds from the fruit peels of L. domesticum and assess their cytotoxic nature against T47D cells. Methods: Powdered peels were macerated with ethyl acetate and the filtrate was evaporated to give EtOAc extract A. Dried extract A was triturated with n-hexane to give n-hexane soluble fraction B and insoluble fraction C. The cytotoxic nature of these three samples were assessed using MTT assay using T47D cells and doxorubicin as a control. Results: Fraction C that showed the smallest IC50 (25.56 ± 0.64μg/mL) value compared to extract A and fraction B. Fraction C was further fractionated by vacuum liquid chromatography to give 6 subfractions. Subfraction 2 showed a single compound based on thin layer chromatography, and this compound was identified as Lamesticumin A on the basis of its spectroscopic data. Lamesticumin A demonstrated cytotoxic activity against T47D cell lines with an IC50 value of 15.68 ± 0.30µg/mL. Conclusions: Further research is needed to investigate the potential of the natural compound Lamesticumin A derived from L. domesticum fruit peel as an anticancer therapy.

Nectin-4 and p95-ErbB2 cooperatively regulate Hippo signaling-dependent SOX2 gene expression, enhancing anchorage-independent T47D cell proliferation

Nectin-4, upregulated in various cancer cells, cis-interacts with ErbB2 and its trastuzumab-resistant splice variants, p95-ErbB2 and ErbB2∆Ex16, enhancing DNA synthesis through the PI3K-AKT signaling in human breast cancer T47D cells in an adherent culture. We found here that nectin-4 and p95-ErbB2, but not nectin-4 and either ErbB2 or ErbB2∆Ex16, cooperatively enhanced SOX2 gene expression and cell proliferation in a suspension culture. This enhancement of T47D cell proliferation in a suspension culture by nectin-4 and p95-ErbB2 was dependent on the SOX2 gene expression. In T47D cells, nectin-4 and any one of p95-ErbB2, ErbB2, or ErbB2∆Ex16 cooperatively activated the PI3K-AKT signaling, known to induce the SOX2 gene expression, to similar extents. However, only a combination of nectin-4 and p95-ErbB2, but not that of nectin-4 and either ErbB2 or ErbB2∆Ex16, cooperatively enhanced the SOX2 gene expression. Detailed studies revealed that only nectin-4 and p95-ErbB2 cooperatively activated the Hippo signaling. YAP inhibited the SOX2 gene expression in this cell line and thus the MST1/2-LATS1/2 signaling-mediated YAP inactivation increased the SOX2 gene expression. These results indicate that only the combination of nectin-4 and p95-ErbB2, but not that of nectin-4 and either ErbB2 or ErbB2∆Ex16, cooperatively regulates the Hippo signaling-dependent SOX2 gene expression, enhancing anchorage-independent T47D cell proliferation.

Cytotoxic Assay of Semipolar Fraction Of Ethanolic Extract From Sugar Apple (Annona Squamosa L.) Stem Bark on T47D Cells

Previous research has shown that some compounds in leaves and seeds of sugar apple have a cytotoxic activity. The aim of this research was to determine the cytotoxicity of semipolar fraction of ethanolic extract from sugar apple stem bark (Annona squamosa L.) on T47D cancer cells. The semipolar fraction of ethanolic extract from sugar apple stem bark was collected by fractionation using Vacuum Liquid Chromatography (VLC) with hexane:ethyl acetic (9:1, 8:2, 7:3, and 6:4) as mobile phase. Cytotoxicity from the fractions of five different concentration namely; 25, 50, 100, 150, and 250, µg/mL was measured by MTT assay. The potency of the cytotoxicity was defined by the ability of the fraction to inhibit the growth of T47D cells indicated by the value of IC50. Qualitative analysis of contained compounds in the fraction was done by Thin Layer Chromatography (TLC) method using silica gel F 254 as a stationary phase and hexane:ethyl acetic (7:3) as a mobile phase. UV 254 and 366 nm lamp also Dragendorff, citroboric, and FeCl3 spray reagents were used to visualize the spots of the secondary metabolites. The result proved that the semipolar fraction of ethanolic extract from sugar apple stem bark showed potential cytotoxicity on T47D cancer cells with IC50value of 70,77 µg/mL. Qualitative analysis showed that the fraction contained flavonoids and alkaloids which is presumably responsible for its cytotoxic activity.