Indonesian Journal of Biotechnology
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Anti-metastatic effect of curcumin analog pentagamaboronon-0-fructose (PGB-0-F) against 4T1 breast cancer cells
No full textDevelopment of a chemotherapeutic agent and boron carrying pharmaceutical based on triple-negative breast cancer is important due to its metastatic progression. Metastases are often more dangerous than the primary tumor and they are responsible for 90% of all cancer deaths. The purpose of this study was to explore the anti-metastatic activities of the PGB-0 complex with fructose (PGB-0-F) against 4T1 breast cancer cells. A scratch wound healing assay was carried out to determine the migration inhibition ability of PGB-0-F, while MMP-9 expression was analysed using gelatin zymography. The testing of anti-migration activity showed that PGB-0-F inhibited in 4T1 cells, whereas the gelatin zymography assay revealed a suppression of MMP-9 expression. PGB-0-F inhibited closure on 4T1 metastatic breast cancer cells line compared with the control. PGB-0-F decreased the MMP-9 expression level compared with the control. Based on these results, PGB-0-F has the potential to be developed as a chemotherapeutic agent, and especially as an anti-metastatic agent
NMR metabolomics revealed metabolites and bioactivity variation in Torbangun leaves Plectranthus amboinicus L. with different origins
No full textPlectranthus amboinicus has been reported to have antidiabetic and antioxidant activities. Environmental factors might influence the plant’s secondary metabolite profile and its beneficial properties. NMR-based metabolomics was used to show phytochemical variations between specimens of P. amboinicus grown in Japan and Indonesia. The results showed that flavonoids and triterpenes were among the discriminating factors of the variation between the two groups. Targeted comparative analysis of the concentration of the specific flavonoids of the plants using a validated HPLC-MWD method showed that the Japanese samples contained a higher concentration of total flavonoids compared with the Indonesian samples. The Japanese and Indonesian samples contained 1100.6 ± 5.1 and 532.4 ± 1.8 µg/g luteolin, and 584.5 ± 7.4 and 571.7 ± 11.6 µg/g apigenin, respectively. Eriodyctiol was detected only in the Indonesian samples. Contrarily, more intensive DPPH reduction and α-glucosidase inhibition activities were found in the Indonesian samples (IC50 14.4 ± 1.2 and 24.0 ± 0.3 µg/mL for the DPPH assay, 1181.9 ± 113.5 and 4451.4 ± 290.0 µg/mL for α-glucosidase inhibition, respectively). Thus, flavonoids might not be the only group of compounds related to the aforementioned bioactivities. This should be confirmed by further research targeting other groups of compounds, such as triterpenes
Cloning and expression of haloacid dehalogenase gene from Bacillus cereus IndB1
No full textOrganohalogen compounds, widely used as pesticides in agriculture and solvents in the industrial sector, cause environmental pollution and health problems due to their toxicity and persistence. Numerous studies have been conducted on the biodegradation of organohalogen compounds, with many focusing on the use of dehalogenase from bacteria. Haloacid dehalogenase is a group of enzymes that cleaves the carbon-halogen bond in halogenated aliphatic acids. In a previous study, the bcfd1 gene encoded haloacid dehalogenase from Bacillus cereus IndB1 was successfully isolated and characterized. This research aimed to create an expression system of the bcfd1 gene by subcloning this gene into pET expression vector and to overexpress the gene in Escherichia coli BL21 (DE3). In addition, the recombinant protein was characterized to gain a better understanding of the catalytic action of this enzyme. A high expression of bcfd1 was obtained by inducing the culture at OD550 0.8–1.0 using 0.01 mM IPTG as determined by SDS-PAGE. Zymogram analysis proved that the recombinant protein possessed dehalogenase activity. Bcfd1 activity toward monochloroacetic acid (MCA) showed specific activity of 37 U/mg at 30°C, pH 9. The predicted tertiary structure of Bcfd1 was estimated has conserved α/ß hydrolase folding motif for haloacid dehalogenase superfamily
In vitro anticancer activity of N-benzyl 1,10-phenanthroline derivatives on human cancer cell lines and their selectivity
No full textThis research was conducted to evaluate the anticancer activity of new compounds of benzyl-1,10- phenanthroline derivatives and their selectivity. In vitro anticancer activity of 11 benzyl-1,10-phenanthroline derivatives were conducted on three human cancer cell lines, cervical cancer (HeLa), myeloma (NS-1), and breast cancer (MCF-7) using MTT-based cytotoxicity assay. The cytotoxicity of each compound was assessed to normal Vero cell line by the same method. The in vitro anticancer activity and cytotoxicity was expressed by the concentration inhibiting 50% of the cell growth (IC50), and the selectivity index (SI) was determined by calculating ratio of the IC50 on Vero cell line and the human cancer cell lines. The results showed that among the 11 compounds tested, the (1)-N-(4-butoxybenzyl)-1,10-phenanthrolinium bromide exhibited the best in vitro anticancer activity with an IC50 27.60 ± 2.76 µM on HeLa, 6.42 ± 5.53 µM on NS-1 and 9.44 ± 2.17 µM on MCF-7 cell lines. Its SI were 377.65 ± 39.97 on HeLa, 6158.72 ± 5306.34 on NS-1 and 1140.11 ± 261.85 on MCF-7 cell lines. This study demonstrated that (1)-N-(4-butoxybenzyl)-1,10-phenanthrolinium bromide possessed a potential in vitro anticancer activity on cancer cell lines with high selectivit
MicroRNA-21 as a biomarker for ovarian cancer detection
No full textOvarian cancer is a lethal disease. One of the problems faced by patients with ovarian cancer is the lack of symptoms in its early stages, which results in it only being detected when it is at an advanced stage. Therefore, there is an urgent need for biomarkers that can predict ovarian cancer precisely. The purpose of this study was to determine the expression of microRNA-21 as a predictive biomarker candidate in both early- and advanced-stage ovarian cancer. This was a cross-sectional study using the blood plasma of 21 healthy control subjects and 37 blood plasma samples from patients with ovarian cancer. Blood plasmas were collected, from which the RNA was isolated. Based on the RNA, the cDNA was synthesized and run through qPCR, the results of which were analyzed using the Livak method. The results showed an upregulation of microRNA-21 in the advanced stage by 2.14 fold compared with the early stage, and 6.13 fold compared with the healthy controls (p < 0.05). The upregulation of microRNA-21 in early-stage ovarian cancer was 2.86 fold compared with the healthy control subjects (p < 0.05). In addition, there was an increase in the expression of microRNA-21 in ovarian cancer by 4.14 fold compared with the healthy controls (p < 0.05). Based on these results, it can be concluded that the expression of microRNA 21 upregulated with the severity of the disease
Agrobacterium tumefaciens-mediated transformation of Jatropha curcas L. with a polyhydroxyalkanoate gene (phaC)
No full textPolyhydroxybutyrate is a component of bioplastics that is synthesized under the control of enzymes encoded by pha multigenes. The genes are naturally present in Ralstonia eutropha. However, the production of bioplastics in bacteria is inefficient because the bacterial biomass is relatively small compared with plants or fungi. As such, engineering techniques have been developed that enable pha genes to be inserted into plant biomass, and then be expressed in the biomass of the plant to produce polyhydroxybutyrate. The objectives of this study were to transform the tissue of Jatropha curcas using the phaC gene (a pha gene), to regenerate the transformed plant, and to confirm the presence of the inserted genes with PCR. The genetic transformation of J. curcas was mediated by Agrobacterium tumefaciens strain GV3101 containing pARTC by dipping the cotyledon tissue of J. curcas in a suspension of the bacterium for 30 min, followed by cocultivation for 3 d on Murashige and Skoog (MS) medium. The tissue was then placed on a selection medium, i.e. MS medium containing 13.3 µM BAP and 0.05 µM IBA with the addition of 20 mg/L kanamycin. The results showed that 12.35% of the tissue survived and regenerated into a shoot after 1–2 months. Molecular analysis of the transformed tissue was performed using phaC and nptII primers, in order to detect the presence of the phaC and nptII genes. Specific bands were detected at 659 bp and 700 bp, corresponding to the nptII primer and phaC primer, respectively
Cytotoxic activity and apoptosis induction of avocado Persea americana Mill. seed extract on MCF-7 cancer cell line
No full textAvocado Persea Americana Mill. is a commercially important crop and studies have shown that the pulp may have benefits to cardiovascular health, dermatological health and possibly anti-cancer activity. Avocado seeds have several medicinal properties such as anti-hyperglycemic, antimicrobial, antioxidant and anti-inflammation. This study aim to evaluate the effect of avocado seed extract on viability and apoptosis of breast cancer cell line MCF-7. The anticancer effect was evaluated by cytotoxic test using MTT assay and the effect on apoptosis and cell cycle was examined by flow cytometry method. The cytotoxic test showed that chloroform extract had strong cytotoxic activity against MCF-7 cell lines with IC50 value of 94.87 µg/mL. Furthermore, the chloroform extract was partitioned with methanol and yield of soluble methanol fraction (FLM) and non soluble methanol fraction (FTLM). The cytotoxic activity of the methanol soluble fraction (FLM) and non soluble methanol fraction (FTLM) against MCF-7 cell lines was increased with IC50 of 34.52 and 66.03 µg/mL, respectively. Flow cytometry analysis using annexin-V and propidium iodide staining revealed that methanol soluble fraction could induce apoptosis and modulating the cell cycle arrest in MCF-7 cell. This research indicated that avocado seed has a potency to induce apoptosis and as anti-proliferative to MCF-7 cells lines
Gelatin extraction from the indigenous Pangasius catfish bone using pineapple liquid waste
No full textGelatin extraction from fish bone has traditionally involved hydrogen chloride and/or sodium hydroxide during pre-treatment. However, these chemicals have begun to be abandoned because of their associated safety and environmental issues. Several studies have looked at the use of citric acid as a safer alternative in fish bone gelatin extraction. The aim of this research was to extract gelatin from the bone of Pangasius catfish with pineapple liquid waste. The extraction was performed in two steps: pre-treatment followed by main extraction at various times (24–56 h) and temperatures (45–75°C). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used as a confirmation test and showed a band for gelatin at ~120 kDa. Gelatin yields were calculated as the ratio of weight of dried gelatin to the total weight of fish ossein. The results indicated that pineapple liquid waste can be used for fish bone gelatin extraction. The recommended conditions for extraction of fish bone gelatin using pineapple liquid waste are 56 h of pre-treatment and 5 h of main extraction at a temperature of 75°C. The gelatin yield was 6.12% and the protein concentration 4.00 g/100 g
Secretory expression of human insulin precursor in Pichia pastoris employing truncated α-factor leader sequence and a short C-peptide
No full textIn the past ten years, diabetes prevalence has increased rapidly in low- and middle-income countries due to lifestyle changes. This increased number of diabetic patients leads to the escalation of recombinant insulin demand, which is creating a large global insulin market. Pichia pastoris has appeared as an alternative host to produce recombinant proteins. It has excellent qualifications as an expression host for large-scale production of recombinant proteins for therapeutic use. In this study, we attempted to express the insulin precursor (IP) in P. pastoris. We used a synthetic IP-encoding gene constructed in frame with the truncated α-factor secretory signal and a short C-peptide (DGK) linked A- and B-chain of human insulin in a pD902 expression vector. Several zeocin resistant clones were successfully obtained and verified with PCR using AOX1 specific primers for the integration of the expression cassette into the P. pastoris genome and for the identification of Mut phenotypes. The secretion of IP by the Pichia pastoris clone in the culture supernatant was confirmed using SDS-PAGE, where a single band of the secreted IP with a molecular mass above 6.5 kDa was found
Bovine vitreous gel can reactivate replicative senescence of human dermal fibroblast
No full textThe most influential factor in the poor healing of chronic ulcers is replicative senescence of fibroblasts that are unresponsive to TGF-β1 stimulation. Cellular replicative senescence can be induced by cultivating normal human dermal fibroblasts (HDFs) in a serum-starved medium. In addition, increasing microenviroment mechanical forces by hyaluronic acid can ameliorate the TGF-β1 signaling of these senescent cells. One of natural resources of hyaluronic acid is bovine vitreous gel. In order to evaluate the effect of bovine-vitreous gel on replicative senescence of fibroblasts, we used various levels of bovine vitreous gel diluted in Dulbecco’s modified Eagle’s medium to stimulate cellular activities of serum-starved HDFs. Those cellular activities were compared to the control media, standardized hyaluronic acid, and to normal HDFs. Our results show that replicative senescence of HDFs treated with 50% bovine vitreous gel exhibited a significantly higher proliferation index, migration rate, and collagen deposition than those cultured in control media, and they displayed an equal level of cellular activity with the HDFs exposed only to standardized hyaluronic acid. We concluded that bovine vitreous gel can be used to stimulate replicative senescence of HDFs and therefore a potential candidate material to stimulate healing of chronic ulcers