Synergistic Effects of Andrographolide on DNA Damage Repair Mechanism and Apoptosis in Breast Cancer Cells

Breast cancer is the second leading cause of cancer deaths in women. Several drugs including cisplatin and carboplatin have shown tremendous effectivity in reducing cancer; however development of drug resistance by breast cancer cells to overcome cytotoxic insults and recurrence of the disease is a major concern at the moment. Andrographolide is a diterpenoid with a potent anti-inflammatory and anti tumor activity and it’s usage in combination therapy would be ideal as it is proven for it’s apoptotic capability in varied number of cells. Antiproliferative and apoptotic activity of andrographolide in triple negative MDA-MB-231 cells was evaluated by clonogenic assay and flow cytometric analysis. Expression and phosphorylation of proteins were evaluated by immunoblotting. Our results revealed dose-dependent cytotoxic effects of andrographolide in MDA-MB-231 cells with and without carboplatin. It resulted in G2/M arrest of cells when treated alone, and further enhanced upon treatment in combination with carboplatin.  Andrographolide alone and in combination with carboplatin enhanced apoptotic cells in early, mid and late stages and increased expression of DNA damage repair response proteins including FANCJ, FANCD2, RAD51, pRPA32 and p53. The present study strongly suggests that andrographolide inhibits breast cancer cell proliferation by apoptosis mediated through cell cycle arrest and up regulation of DNA damage repair response gene expression and shows synergistic effects upon usage in combination with carboplatin

Evaluation of TNF-α, IL-10 and IL-6 Cytokine Production and Their Correlation with Genotype Variants amongst Tuberculosis Patients and Their Household Contacts.

BackgroundHousehold contacts of diagnostically established tuberculosis (TB) patients are highly susceptible to disease development. It is surmised that cytokines perhaps play a synergistic and a prognostic role in the activation of the otherwise latent infection in these house hold contacts. Evaluation of the cytokines and any of their inherent polymorphisms might provide a useful diagnostic tool in evaluating the immune regulation and the progression of the disease. The cytokines thus released in a paracrine manner in serum may also provide an indirect measure of the cytokine function.ObjectiveThe present study was aimed to evaluate the levels of TNF-α, IL-10 & IL-6 cytokines and their correlation with genotype variants amongst tuberculosis patients and their household contacts.MethodsThe cytokine levels were estimated in serum by enzyme-linked immunosorbent assay (ELISA) and their polymorphisms were studied by amplification refractory mutation system polymerase chain reaction (ARMs PCR) in active pulmonary tuberculosis patients (APTB = 150), household contacts (HHC = 190), and healthy controls (HC = 150).ResultsThe median values of TNF-α cytokine were significantly high among APTB and HHC compared to HCs (PConclusionLarge sample size with follow-up at different time points may further illuminate the role of IL-10 and IL-6 cytokines as a prognostic marker in house hold contacts

Comparison of cytokines in APTB, HHC and HC categories.

<p>(a) Ratio of TNF-α/IL-10 serum levels (b) Ratio of IL-6/IL-10 serum levels. Results are represented as box plot. The threshold for significance was set at p ≤ 0.05. Bars above the plots represent the statistical differences between the groups.</p

TNF-α (-308A/G), IL-10 (-1082G/A), IL-6 (-174G/C) gene polymorphism.

<p>(1a) Lane 1–100 bp ladder;Lane 2–3;4–5;6–7;8-912-13-GA genotype; 10-11-GG genotype; respectively.(1b) Lane 1 – 100bp Ladder; Lane 2 &3 to 8& 9 –GA genotype; Lane 10&11 to 12&13 –GG genotype.(1c) Lane 100- 1000bp ladder;Lane 2-3-GC; 4-5-GC; 6–7,8-9-GG;10-11-CC;12-13-CC genotype.</p

ROC plots for IL-10 and IL-6 for the comparison between patient (APTB) and control (HC) groups.

<p>ROC plots for IL-10 and IL-6 for the comparison between patient (APTB) and control (HC) groups.</p

Serum levels of TNF-α, IL-10, IL-6.

<p>(2a, 2b, 2c) Data are shown as box plots, where the boxes represent the first through third quartiles, the lines within the boxes represent the median, and the lines outside the boxes represent the minimum and maximum values (excluding outliers). APTB- active pulmonary Tuberculosis patients; HHC-household contacts; HC-healthy controls.</p

Comparision of cytokine gene polymorphisms of TNF-α (-308A/G), IL-10(-1082G/A) and IL-6(-174G/C) with serum levels.

<p>(4a, 4b, 4c) Vertical axis- serum levels showing median values; Horizontal axis- genotypes of TNF- α (-308A/G); IL-10(-1082G/A) and IL-6(-174G/C),APTB—active pulmonary tuberculosis patients; HHC-household contacts; HC-healthy controls.</p

Clinical characteristic of APTB, HHC and HC.

<p><b>*</b> P value by chi-square analysis;</p><p>^#–significance by T Test</p><p>ns- not significant;</p><p>NA–not applicable.</p><p>Clinical characteristic of APTB, HHC and HC.</p

Comparison of TNF- α, IL-10 and IL-6 between patient (APTB) and Control (HC) groups.

<p>*- significant p<0.05</p><p>AUC- area under the curve.</p><p>Comparison of TNF- α, IL-10 and IL-6 between patient (APTB) and Control (HC) groups.</p