ALTERNATIVE SPLICING OF mRNA TRAIL REGULATES APOPTOSIS IN THE GLIOBLASTOMA MULTIFORME T-98G CELL LINE

Objective: This is an in vitro experimental study designed to analyze the role of alternative splicing of mRNA in the apoptotic process of the cancer cells. Here we induced apoptosis in the glioblastoma multiforme (GBM) T-98G cell line to obtain a better understanding in the regulation of mRNA expression of the soluble Tumor Necrosis factor-related Apoptosis-Inducing Ligand (sTRAIL) gene. Methods: Cells were induced to undergo apoptosis by treatment with rotenone at 10, 20 and 40 µM for 6 h. Dimethylsulphoxide (DMSO) was used to dissolve rotenone and as a negative control. The morphology of the GBM-T98G cells was viewed with an inverted microscope. DNA, RNA and protein extractions were performed to analyse apoptotic DNA fragmentation by a DNA laddering assay, a quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) for TRAIL mRNA expression and ELISA for caspase-9 protein expression. Electrophoresis was also performed on TRAIL complementary DNA (cDNA) produced from TRAIL qRT-PCR mRNA. Results: Nucleosomal DNA degradation was confirmed by DNA laddering, whereas the TRAIL melting curve and the cDNA electrophoresis showed a shift in the balance of the TRAIL mRNA isoform to the pro-apoptotic mRNA isoform, in conjunction with a significant increase in expression of TRAIL mRNA and caspase-9 protein. Conclusion: These findings indicate the regulation of apoptotic events at the level of TRAIL mRNA expression, as indicated by the shift in the balance of mRNA expression of the TRAIL isoform towards the pro-apoptotic isoform

Construction of recombinant sox2-encoding plasmids that regulate pluripotency of breast cancer stem cells from indonesian patient

A therapy development with recombinant protein is a new and potential innovation to destroy cancer stem cells (CSCs). Various ways of killing CSCs include the provision of polyvalent antiprotein antibodies that code pluripotency. Therefore, it takes a mixture of Oct-4, c-Myc, Sox2, and Klf4 protein antigens that can stimulate the formation of polyvalent antibodies. This study aimed to construct Sox2 recombinant by identifying the target genes by reverse transcriptase PCR and then arranging their designs to be inserted into the cloning vector pET101/D-TOPO®. The target gene was developed by finding the complete sequences of Sox2 nucleotides on the NCBI GenBank. The growth on LB-ampicillin agar plates was amplified by PCR to obtain colonies with pET101/D-TOPO® vectors and inserts of the pluripotent gene of CSCs, then the PCR results were observed through electrophoresis. A total of fifteen colonies have DNA bands with a base pair of about 300 bp in length. The recombinant clones produced Sox2 genes with a base length of 330 bp

HDAC2 and PCNA expression is correlated to decreasing of endoxifen sensitivity in human breast cancer stem cells ALDH+

Latar belakang: Sel punca kanker payudara (breast cancer stem cells/BCSC) adalah subpopulasi sel kanker yang memiliki kemampuan menghasilkan tumor baru dan bersifat seperti sel punca. Penelitian kami sebelumnya menggunakan jaringan kanker payudara mengungkapkan bahwa ekspresi gen histone deacetylase 2 (HDAC2) dan proliferating cell nuclear antigen (PCNA) ditemukan perbedaan signifikan setelah terapi neoajuvan hormon dan kemoterapi. Penelitian ini bertujuan untuk menganalisis hubungan antara ekspresi HDAC2 dan PCNA dengan kelangsungan hidup sel punca kanker payudara dengan penanda aldehyde dehydrogenase + (ALDH+) yang diberi perlakuan endoksifen. Metode: Sampel adalah BCSC primer manusia ALDH+ yang diberi perlakuan endoksifen 4 uM masingmasing selama 2, 4, 6, 8, 10, 12, 14 hari. Viabilitas sel dilihat dengan menggunakan trypan blue dan ekspresi mRNA HDAC2 dan PCNA ditentukan menggunakan qRT-PCR. Hasil: Viabilitas BCSCs ALDH + menurun setelah 2 sampai 4 hari pemberian endoksifen. Pada periode ini juga didapatkan ekspresi mRNA HDAC2 dan PCNA mengalami penurunan. Tetapi setelah pemberian endoksifen selama 8 hari, viabilitas BCSCs ALDH + mengalami peningkatan dan ditemukan peningkatan yang signifikan pada hari ke-14 pemberian endoksifen. Ekspresi mRNA HDAC2 dan PCNA juga menunjukkan peningkatan mulai pada hari ke-8 dan terus meningkat hingga hari ke-14 pemberian endoksifen. Penelitian ini menunjukkan pola yang sama antara ekspresi mRNA HDAC2 dan PCNA dan viabilitas sel. Kesimpulan: Induksi endoksifen yang lama menurunkan sensitivitas efek endoksifen pada BCSC manusia dan ekspresi HDAC2 dan PCNA berkorelasi dengan viabilitas BCSC manusia setelah induksi endoksifen. (Health Science Journal of Indonesia 2019;10(2):77-81) Kata kunci: sel punca kanker payudara, viabilitas sel, HDAC2, PCNA, endoksifen   Abstract Background: Breast cancer stem cells (BCSCs) are subpopulation of cancer cells that has the ability to generate new tumor and similar properties to stem cell. Our previous study using breast cancer patients revealed that gene expression of histone deacetylase 2 (HDAC2) and proliferating cell nuclear antigen (PCNA) were significantly altered after neoadjuvant hormone and chemotherapy. This study aimed to analyze the correlation between HDAC2 and PCNA expressions with the viability of breast cancer stem cells aldehyde dehydrogenase + (BCSC ALDH+) treated by endoxifen. Method: Samples are human primary BCSCs ALDH+ that treated with 4 uM of endoxifen for 2, 4, 6, 8, 10, 12, 14 days, respectively. Cell viability was measured using trypan blue exclusion assay and the mRNA expressions of HDAC2 and PCNA were determined using qRT-PCR. Results: The viability of BCSCs ALDH+ was decreased after 2 days until 4 days-endoxifen treatment. It also demonstrated that mRNA expression of HDAC2 and PCNA were decreased in this period. But after 8 days endoxifen treatment, the viability of BCSCs ALDH+ was increased. The increasing of viability was higher in 14 days-endoxifen treatment. The mRNA expression of HDAC2 and PCNA also showed increasing begin on 8 days and continued to increase until 14-days endoxifen treatment. We found a similar pattern between HDAC2 and PCNA expression and cell viability. Conclusion: Prolonge endoxifen treatment decrease sensitivity of endoxifen effect in human BCSC and the expression of HDAC2 and PCNA are correlated to human BCSCs viability after endoxifen treatment. (Health Science Journal of Indonesia 2019;10(2):77-81) Keywords: human breast cancer stem cells, viability, HDAC2, PCNA, endoxife

PIPERINE IN THE PREVENTION OF THE DECREASED TAMOXIFEN SENSITIVITY IN MCF-7 BREAST CANCER CELL LINE

Objective: This study was designed to analyze the role of piperine in modulating P-glycoprotein mRNA expression when added in combination withtamoxifen to breast cancer cells in culture.Methods: MCF-7 breast cancer cells were treated with 1 μM tamoxifen with or without piperine (12.5, 25, or 50 μM) or verapamil 50 μM (P-glycoproteininhibitor positive control) for up to 12 days. We assessed the cell viability and isolated total RNA from them. We quantified P-glycoprotein expressionsusing quantitative reverse transcription polymerase chain reaction.Results: Administration of various doses of piperine decreased MCF-7 breast cancer cell viability. Piperine, when given in combination with tamoxifen,decreased the expression of P-glycoprotein mRNA in cells compared with the expression in cells treated with tamoxifen only. The effects were shownto be dose dependent.Conclusion: Piperine prevents the development of breast cancer cell tamoxifen resistance, probably through its inhibition of P-glycoprotein expression

CURCUMIN INCREASES THE SENSITIVITY OF BREAST CANCER CELLS TO TAMOXIFEN BY INHIBITING MRP2 MRNA EXPRESSION OF EFFLUX TRANSPORTER MRP2

Objective: Tamoxifen is the drug of choice to treat breast cancer positive for estrogen receptor. Long-term use of tamoxifen can induce multidrug resistance (MDR) associated with decreased sensitivity of cancer cells to the drug. One of the causes of MDR is overexpression of efflux transporter multidrug resistance-associated protein (MRP)2. Various drugs are known to act as MRP2 inhibitors, including curcumin. This study investigated the effects of curcumin on the sensitivity of breast cancer cells to tamoxifen through inhibition of MRP2. Methods: We used MCF-7 cells that were previously exposed to long-term tamoxifen treatment [MCF-7(T) cells]. MCF-7(T) cells were treated with 1 µM tamoxifen, curcumin (5, 10, and 20 µM), combinations of curcumin (5, 10, and 20 µM) and 1 µM tamoxifen, or 10 µM nevirapine (a known MRP2 inhibitor) for 5 d. Then, the cells were harvested, counted to assess cell viability, and evaluated for MRP2 mRNA expression. Results: Treatment with curcumin alone or in combination with tamoxifen significantly reduced cell viability at all curcumin concentrations compared with the control. The reduction in cell viability was accompanied by a reduced level of MRP2 mRNA expression. Conclusion: Application of curcumin to MCF-7 cells previously exposed to long-term tamoxifen treatment increase the sensitivity of cancer cells to tamoxifen. The increased sensitivity of these cells was attributed, at least in part, to inhibition of the efflux transporter MRP2

Efek Hipoxia Mimetic Cobalt Chloride (CoC12 )terhadap Ekspresi mRNA dan Aktivitas Spesifik Manganese Superoksida Dismutase (Mn SOD ) Ginjal Tikus

MnSOD is a primary antioxidant that protects cells from oxidative stress due to H2O2 inmitochondrial membranes. This study aims to determine the effect of CoCl2 as hypoxic mimetic agent on mRNA expression of MnSOD and MnSOD-specific activity in rat kidney. Male Sprague dawley rat are induced with 30mg/Kg body weight CoCl2 intraperitoneally. After that, the experiment was divided into 3 groups: 2, 8, and 24 hours incubation after injection. All groups are compared with control group without injection. All of rat were terminated and the kidneys were removed. mRNA expression and specific activity of the kidney MnSOD are measured. After 2 hours, mRNA expression increased up to 42 times, after 8 and 24 hours return to normal. Specific activity of MnSOD in 2 hours after injection has not changed yet, and after 8 and 24 hours increased 1.5 times. This study shows that induction of Hipoxic mimetic CoCl2 enhances mRNA expression and MnSOD-specific activity

Drug Efflux Transporters Are Overexpressed in Short-Term Tamoxifen-Induced MCF7 Breast Cancer Cells

Tamoxifen is the first line drug used in the treatment of estrogen receptor-positive (ER+) breast cancer. The development of multidrug resistance (MDR) to tamoxifen remains a major challenge in the treatment of cancer. One of the mechanisms related to MDR is decrease of drug influx via overexpression of drug efflux transporters such as P-glycoprotein (P-gp/MDR1), multidrug resistance associated protein (MRP), or BCRP (breast cancer resistance protein). We aimed to investigate whether the sensitivity of tamoxifen to the cells is maintained through the short period and whether the expressions of several drug efflux transporters have been upregulated. We exposed MCF7 breast cancer cells with tamoxifen 1 μM for 10 passages (MCF7 (T)). The result showed that MCF7 began to lose their sensitivity to tamoxifen from the second passage. MCF7 (T) also showed a significant increase in all transporters examined compared with MCF7 parent cells. The result also showed a significant increase of CC50 in MCF7 (T) compared to that in MCF7 (97.54 μM and 3.04 μM, resp.). In conclusion, we suggest that the expression of several drug efflux transporters such as P-glycoprotein, MRP2, and BCRP might be used and further studied as a marker in the development of tamoxifen resistance

The Sensitivity of Human Breast Cancer Stem Cells (ALDH+) Against Doxorubicin Treatment is Associated with PCNA and BIRC5 Gene Expressions

Introduction: Breast cancer stem cells (BCSCs) are identified as side populations in breast cancer cells owing stem cell properties and tumorigenic characteristics. Previous studies revealed that breast cancer chemotherapy led to BCSC enrichment which contributed to therapy resistance. Our recent in vivo study using Next Generation Sequencing has demonstrated that PCNA - the proliferative gene - and BIRC5 – the anti-apoptosis gene – were under-expressed in human breast tumors after neoadjuvant chemotherapy. This study aimed to verify the role of PCNA and BIRC5 expression in doxorubicin-treated human BCSCs in vitro and its association with cell viability. Method: Human BCSCs (ALDH+) were treated with 0.25 uM of doxorubicin for 2, 4, 6, 8, 10, 12, 14 days respectively. Cell viability was measured using trypan blue exclusion assay and the expressions of PCNA and BIRC4 mRNA were determined using qRT-PCR. Results: This study demonstrated that the viability of ALDH+ BCSCs decreased after 2 days and increased again after 8 days of doxorubicin treatment, indicating the decrease of doxorubicin sensitivity. Interestingly, PCNA and BIRC5 genes were modulated in line with the modulation of cell viability during doxorubicin treatment of human BCSCs. Conclusion: In conclusion, we suggest that the PCNA and BIRC5 expressions play an important role on the BCSCs viability which associated with the sensitivity of doxorubicin treatment

Myoglobin Expression in Chelonia mydas Brain, Heart and Liver Tissues

An understanding of the underpinning physiology and biochemistry of animals is essential to properly understand the impact of anthropogenic changes and natural catastrophes upon the conservation of endangered species. An observation on the tissue location of the key respiratory protein, myoglobin, now opens up new opportunities for understanding how hypoxia tolerance impacts on diving lifestyle in turtles. The respiratory protein, myoglobin has functions other than oxygen binding which are involved in hypoxia tolerance, including metabolism of reactive oxygen species and of the vascular function by metabolism of nitric oxide. Our work aims to determine whether myoglobin expression in the green turtle exists in multiple non muscle tissues and to confirm the hypothesis that reptiles also have a distributed myoglobin expression which is linked to the hypoxiatolerant trait. This initial work in turtle hatch Chelonia mydas confirms the presence of myoglobin transcriptin brain, heart and liver tissues. Furthermore, it will serve as a tool for completing the sequence and generating an in situ hybridization probe for verifying of cell location in expressing tissues