Resveratrol modulates expression of ABC transporters in non-small lung cancer cells: Molecular docking and gene expression studies 1 S. KARTHIKEYAN and S.L. HOTI Regional Medical Research Centre (ICMR), Nehru Nagar , Belgaum, Karnataka, India.

L ung cancer Comprising 17% of the total new cancer cases and 23% of the total cancer deaths in males. Among females, lung cancer is the fourth most commonly diagnosed cancer. Among males, cancers of sites associated with use of tobacco are the most frequent. BACKGROUND Lung Cancer Fact Sheet,

ESTIMATED NEW CANCER CASES & DEATHS, 2014 One million lung cancer deaths were observed as per 2012 survey. In 2020, the figure is projected at 1.5 million. Carbone, 1997 ACS. Cancer Facts and Figures, 2014Cancer Facts and Figures,

One of the frequent tumors in the elderly. Constitutes 75-80% of all lung cancers. Late diagnosis of the tumor. NSCLC cells are relatively resistant to chemo-radiotherapy. NON SMALL CELL LUNG CANCER (NSCLC) 4

Cancer Multidrug Resistance Cancer cells resist treatments with anticancer drugs. A major mechanism of cancer multidrug resistance is the reduced accumulation of drugs due to decreased uptake and increased efflux. 5

ATP-binding cassettes (ABC) drug transporters Decreased drug uptake Increased drug efflux TMD NBD ABC transporters are implicated in the development of multidrug resistance (MDR) in cancer cells. P-glycoprotein (P-gp; ABCB1), Multidrug resistant-associated protein (MRP1; ABCC1) Breast cancer resistant protein (BCRP; ABCG2; MXR). 6

ABC inhibitors as chemosensitizers The first three generation ABC inhibitors (varapamil, cyclosporin-A, tariquitor) shows undesirable side effects. Unfortunately, progress in finding a potent, selective P-gp inhibitor to modulate ABC transporters and restore drug sensitivity in multidrug-resistant cancer cells has been slow and challenging. Candidate drug should ideally be selective, potent and relatively non-toxic. Utilizing natural products for the development of the next generation inhibitors will be a novel approach. 7

Resveratrol Resveratrol (RSV) naturally occurring polyphenolic phytoalexin Synthesized by a grapes, berries and peanuts etc. Its beneficial effects against many diseases such as cancer, cardiovascular disease, inflammatory disease, and platelet aggregation. Trans-3,4',5-trihydroxystilbene (C 14 H 12 O 3 ), Mol. wt

(a)(b) (c) (d) Molecular interaction of RSV (CID:445154) with TMD region of P-gp (PDB ID: 3G61) The values of docking score, glide score and hydrogen bond score indicates RSV possess binding affinity with TMD region. Thereby, it may inhibit ABC transporters function. Hence, in this study, we have chosen RSV as ABC inhibitor to enhance paclitaxel efficacy. 9 Docking scoreGlide scoreHydrogen bond score

Paclitaxel Isolated from Taxes brevifolia. Potent anticancer agent. Paclitaxel binds to the β subunit of tubulin. Blocking cells at late G2 mitotic phase of cell cycle. Regular PTX administration develops MDR in lung cancer patients. Paclitaxel (MW ) Taxes brevifolia 10

Chemosensitizing potential of Resveratrol in NCI-H460 cells Study model: NCI-H460 was obtained from NCCS, Pune. These cell lines are developed from NSCLC. NSCLCs cells are relatively insensitive to chemotherapy. Hence, NCI-H460 was chosen for the cancer multidrug resistance study. 11 Photographs shows morphology of PTX resistant NSCLC cell line

Fig. 1 Selection of optimum doses of RSV and PTX IC 50/24 h = 10 µg/mL IC 50/24 h = 5 µg/mL (A)(B) Effect of (A) RSV and (B) PTX on NCI-H460 cytotoxicity upon 24 h incubation period. Values are given as means  S.D. of six experiments in each group. 12

13 Resveratrol + Paclitaxel ABC transporters expression Cell cycle progression Membrane transport function Experimental Scheme Cytotoxicity, ROS generation and Apoptotic morphological changes 13 NCI-H460 cells were treated with RSV 1 h before PTX exposure and incubated for 24hr Control RSV (10 µg/ml) PTX (5 µg/ml) RSV (10 µg/ml) + PTX ( 5 µg/ml)

Control RSV (10 µg/ml) PTX (5 µg/ml) RSV (10 µg/ml) +PTX (5 µg/ml) Fig. 2 Effect of RSV, PTX and RSV-PTX on cytotoxicity in NCI-H460 cells Microscopic images show the purple colored crystal formation, which is an indication of cell viability Values are given as means  SD of six experiments in each group. Values not sharing a common marking (a, b, c,..) differ significantly at P ≤ 0.05 (DMRT). (A)(B) 14

Control RSV PTX RSV - PTX NCI-H460 cells were incubated with RSV, PTX and RSV-PTX. The level of fluorescence intensity of rhodamine123 was analyzed by flow cytometry Fig. 3 Effect of RSV-PTX on membrane transport function in NCI-H460 cells Inhibitor 15

Fig. 4 Effect of RSV-PTX on cell cycle progression in NCI-H460 cells Control RSV (10 µg/ml ) PTX (5 µg/ml ) RSV (10 µg/ml ) +PTX (5 µg/ml ) Effect of RSV-PTX on cell cycle and apoptosis (a) NCI-H460 cells were treated with RSV, PTX or RSV-PTX. Only RSV + PTX were able to increase the proportion of cells in G2/M as compared to RSV alone or PTX alone treated cells. (b) The bar graphs show the cell cycle distribution and the percentage of cells in each phase of the cell cycle. Percentage of total cells was obtained by using the CellQuest software. a b 16

A. Western blot analysisB. Band intensity by densitometry Lane 1: Control;Lane 2: RSV; Lane 3: PTX;Lane 4: RSV +PTX Lanes P-gp β -actin Fig. 5 Effect of RSV-PTX on the expression of P-gp in NCI-H460 cells The graph represents the P-gp quantification values normalized to β-actin levels 17

ABCB1/ MDR1 ABCB4/ MDR3 Fig. 6 Effect of RSV-PTX on mRNA expression patterns of ABCB1 and ABCB4 in NCI-H460 cells A. mRNA expression by qRT-PCR B. relative gene expression The graph represents the quantification results normalized to 18S rRNA 18

Fig. 7 Effect of RSV-PTX on mRNA expression patterns of LRP and ABCC1 in NCI-H460 cells A. mRNA expression by qRT-PCR B. Relative gene expression LRP/ MVP ABCC1/ MRP1 The graph represents the quantification results normalized to 18S rRNA 19

Fig. 8 Effect of RSV-PTX on mRNA expression patterns of ABCC2 and ABCC3 in NCI-H460 cells A. mRNA expression by qRT-PCR B. Relative gene expression ABCC2/ MRP2 ABCC3 /MRP3 The graph represents the quantification results normalized to 18S rRNA 20

1.RSV-PTX combination show superior anti-proliferation effect in NCI-H460 cells. 2.RSV treatment inhibit ABC transporters function and thereby may enhance more PTX accumulation inside the NCI-H460 cells. 3.RSV-PTX combination enhances G2/M apoptotic cell death. 4.RSV, PTX and RSV-PTX combination down-regulates various ABC transporters expression in NCI-H460 cells Conclusion 21

Recommendations The RSV-PTX combination showed better cell death efficacy. This may be due to the additive or synergistic effect of RSV with PTX. 22 A number of additional experiments are underway. The anticancer efficacy of RSV and PTX combination will be tested in human tumor xenograft bearing BALB/c nude mice. NCI-H460 cells grown in vitro will be implanted sub-cuataneously under the shoulder in the BALB/c nude mice. Tumor volume, body weight, Ex vivo tissue imaging and immunofluorescence studies will be employed to understand the tumour regression in RSV-PTX treated animals Future directions

23 ACKNOWLEDGEMENTS This work was supported by research grant from the Indian Council of Medical Research (ICMR), Ministry of Health and Family Welfare, Government of India, New Delhi to Dr. S. Karthikeyan.