Evaluation of Anticancer Activity of Curcumin Analogues Bearing a Heterocyclic Nucleus.

We report herein an in vitro anticancer evaluation of a series of seven curcumin analogues (3a-g). The National Cancer Institute (NCI US) Protocol was followed and all the compounds were evaluated for their anticancer activity on nine different panels (leukemia, non small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and breast cancer) represented by 60 NCI human cancer cell lines. All the compounds showed significant anticancer activity in one dose assay (drug concentration 10 μM) and hence were evaluated further in five dose assays (0.01, 0.1, 1, 10 and 100 μM) and three dose related parameters GI50, TGI and LC50 were calculated for each (3a-g) in micro molar drug concentrations (μM). The compound 3d (NSC 757927) showed maximum mean percent growth inhibition (PGI) of 112.2%, while compound 3g (NSC 763374) showed less mean PGI of 40.1% in the one dose assay. The maximum anticancer activity was observed with the SR (leukemia) cell line with a GI50 of 0.03 μM. The calculated average sensitivity of all cell lines of a particular subpanel toward the test agent showed that all the curcumin analogues showed maximum activity on leukemia cell lines with GI50 values between 0.23 and 2.67 μM.


Evaluation of Anticancer Activity of Curcumin Analogues Bearing a Heterocyclic Nucleus
Mohamed Jawed Ahsan new and better chemotherapeutic agents. Curcumin, a β-diketone is one of the active ingredients obtained from the powdered root of Curcuma longa Linn., which showed a wide spectrum biological activity. The four different sites (aryl side chain, diketo group, double bonds, and active methylene group) of curcumin were exploited and a large no of semi-synthetic analogues as well as synthetic analogues of curcumin was prepared by medicinal chemists with significantly improved biological activity (Vyas et al., 2013;Balaji et al., 2015).
Curcumin itself is more toxic towards cancerous cells as compared to the normal human cell, (Kunwar et al., 2008). It can be concluded that curcumin analogues would have more specificity towards the cancer cells. In another study, curcumin showed autophagic and apoptotic death of K562 cell line (leukemia) (Jia et al., 2009). Several curcumin analogues were reported as anticancer agent (Sharma et al., 2015;Ahsan et al., 2013;Liang et al., 2009). Apart from anticancer activity various other activity were also reported that includes antibacterial (Lal el al., 2012;Sahu et al., 2012;Zhichang et al., 2012), anti-HIV (Singh et al., 2010), anti-inflammatory (Saja et al., 2007), antimalarial Balaji et al., 2015), anti-EGFR activity (Ahsan et al., 2013;Yadav et al., 2014).and many more have been reported for curcumin analogues. These recent development, made curcumin as an ideal lead compound for future drug discovery. The promising anticancer activity of curcumin inspired us to explore curcumin analogues further. The present investigation is the continuation of the previous work in which the anticancer activity was reported on 60 NCI cancer cell lines at 10 µM drug concentration (one dose assay) (Ahsan et al., 2013). And we reported herein the anticancer activity of curcumin analogues in five dose assay (0.01, 0.1, 1, 10 and 100 µM) and three dose related parameters GI 50 , TGI and LC 50 were calculated for each tested compounds (3a-g) in micro molar drug concentrations (µM).

Chemistry
All the chemicals were supplied by Merck (Germany), Konark Herbal (India) and S. D. Fine Chemicals (India). Melting points were determined by open tube capillary method and are uncorrected. IR spectra were obtained on a Schimadzu 8201 PC, FT-IR spectrometer (KBr pellets). 1H NMR spectra were recorded on a Bruker AC 400 MHz spectrometer using TMS as internal standard in DMSO d6. Mass spectra were recorded on a Bruker Esquire LCMS using ESI and elemental analyses were performed on Perkin-Elmer 2400 Elemental Analyzer.

Cancer cell lines
The antiproliferative activity of the was carried out at National Cancer Institute (NCI US) on nine different panels viz. leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate, and breast cancers of nearly 60 cancer cell lines (60 NCI cancer cell lines).

Anticancer activity
The anticancer screening was carried out on leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate, and breast cancers cell lines, nearly 60 in number according to the reported NCI US protocol (http://dtp.nci.nih.gov; Boyd et al., 1995;Monks et al., 1991;Shoemaker 2006). Using the seven absorbance measurements [time zero, (Ti), control growth, (C), and test growth in the presence of drug at the five concentration levels (Tf)], the percentage growth was calculated at each of the drug concentrations levels as: [(Tf-Ti)/(C-Ti)] x 100 for concentrations for which Tf ≥ Ti and [(Tf-Ti)/Ti] x 100 for concentrations for which Tf < Ti (http://dtp.nci.nih.gov; Ahsan et al., 2013). Three-dose response parameters (GI 50 , TGI, and LC 50 ) were calculated for each of the experimental agents in five dose assay. Growth inhibition of 50% (GI 50 ) was calculated from 100 × [(Tf-Ti)/(C-Ti)] = 50, which was the drug concentration resulting in a 50% reduction in the net protein increase (as measured by sulforhodamine B, SRB staining) in control cells during the drug incubation. The total growth inhibition (TGI) was calculated from Tf = Ti, which was the drug concentration resulting in total growth inhibition and signified the cytostatic effect. The LC 50 was calculated from 100 × [(Tf-Ti)/Ti] = -50, indicating a net loss of cells following treatment which indicated the concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning (Grever et al., 1992;Alley et al., 1998;Corona et al., 2009;Ahsan et al., 2013).
Three-dose response parameters (GI 50 , TGI, and LC 50 ) were calculated for each of the experimental agents. Growth inhibition of 50% (GI 50 ) was calculated from 100 × [(Tf-Ti)/(C-Ti)] = 50, which was the drug concentration resulting in a 50% reduction in the net protein increase (as measured by sulforhodamine B, SRB staining) in control cells during the drug incubation. The total growth inhibition (TGI) was calculated from Tf = Ti, which was the drug concentration resulting in total growth inhibition and signified the cytostatic effect. The LC 50 was calculated from 100 × [(Tf-Ti)/Ti] = -50, indicating a net loss of cells following treatment which indicated the concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning. Values were calculated for each of these three parameters at the level of activity; however, if the effect did not reach to the level of activity, the value of parameter was expressed as less than the minimum concentration tested, or if the effect exceeded the level of activity, the value of parameter was expressed as greater than the maximum concentration tested (http://dtp.nci.nih.gov; Alley et al., 1988;Grever et al., 1992;Ahsan et al., 2013). LogGI 50 , log TGI, and log LC 50 are the logarithm molar concentrations producing 50% growth inhibition (GI 50 ), a total growth inhibition (TGI), and a 50% cellular death (LC 50 ), respectively.

Anticancer activity
The curcumin analogues (3a-g) showed promising  DOI:http://dx.doi.org/10.7314/APJCP.2016.17.4.1739 Evaluation of Anticancer Activity of Curcumin Analogues Bearing a Heterocyclic Nucleus anticancer activity in single dose assay with mean percent growth inhibition (PGI) ranging between 112.2 and 40.1 percent (Ahsan et al., 2013). The compound 3d showed maximum anticancer activity with PGI of 112.2%, while compound 3g showed less anticancer activity with PGI of 40.1% (Figure 1). All the compounds (3a-g) was found to be active and met the pre-determine criterion of growth inhibition and thus further chosen for the NCI full panel of five dose assay at five different drug concentrations (0.01, 0.1, 1, 10 and 100 µM). Three-dose response parameters (GI 50 , TGI, and LC 50 ) were calculated for each of the experimental agents in five dose assay is given in Table 1. The compound 3a showed highest sensitivity to SR (leukemia) with GI 50 of 0.03 µM and least sensitivity to OVCAR-5 (ovarian cancer) with GI 50 of 3.30 µM. The best value of TGI was being noted on SR (leukemia) with 0.09 µM. Only in 15 cell lines the compound 3a registered LC 50 value of >100 µM. The compound 3b showed highest sensitivity to SR (leukemia) with GI 50 of 0.06 µM and least sensitivity to HT29 (colon cancer) with GI 50 of 2.52 µM. The best value of TGI was being noted on MDA-MB 435 (melanoma) with 0.37 µM. Only in 24 cell lines the compound 3b registered LC 50 value of >100 µM. The compound 3c showed highest sensitivity to MDA-MB 435 (melanoma) with GI 50 of 0.23 µM and least sensitivity to SW620 (colon cancer) with GI 50 of 54.90 µM. The best value of TGI was being noted on MDA-MB 435 (melanoma) with 0.57 µM. Only in 42 cell lines the compound 3c registered LC 50 value of >100 µM. The compound 3d showed highest sensitivity to SR (leukemia) with GI 50 of 0.04 µM and least sensitivity to COLO205 (colon cancer) with GI 50 of 1.73 µM. The best value of TGI was being noted on SR (leukemia) with 0.29 µM. Only in 24 cell lines the compound 3d registered LC 50 value of >100 µM. The compound 3e showed highest sensitivity to SR (leukemia) with GI 50 of 0.03 µM and least sensitivity to OVCAR-5 (ovarian cancer) with GI 50 of 2.67 µM. The best value of TGI was being noted on SR (leukemia) with 0.15 µM. Only in 24 cell lines compound 3e registered LC 50 value of >100 µM. The compound 3f showed highest sensitivity to SR (leukemia) with GI 50 of 0.34 µM and least sensitivity to EKVX (non small cell lung cancer) with GI 50 of 4.72 µM. The best value of TGI was being noted on RXF (renal cancer) with 3.96 µM. Nearly on 57 cell lines compound 3f registered LC 50 value of >100 µM. The compound 3g showed highest sensitivity to HT29 (colon cancer) with GI 50 of 1.30 µM and least sensitivity to NCI ADR-RES (ovarian cancer) with GI 50 of 16.7 µM. The best value of TGI was being noted on HCT-116 (colon cancer) with 1.24 µM. Nearly on 50 cell lines compound 3g registered LC 50 value of >100 µM. All the curcumin analogues showed promising anticancer activity with GI 50 between 0.03 µM (SR; leukemia) and 54.9 µM (SW620; colon cancer). Further average sensitivity on a particular panel of cell lines was calculated for each compounds (3ag) showed a relatively higher sensitivity towards leukemia   DOI:http://dx.doi.org/10.7314/APJCP.2016.17.4.1739 Evaluation of Anticancer Activity of Curcumin Analogues Bearing a Heterocyclic Nucleus cell lines with average GI 50 value ranging between 0.24 and 2.67 µM ( Table 2). The average sensitivity of each compound (3a-g) on a particular panel of cell lines is shown in Figure 2. All these curcumin analogues showed comparatively higher activity than curcumin except for the compound 3c which showed maximum GI 50 of 15.3 µM on colon cancer. The anticancer data of curcumin in Table 2 and Figure 2 were taken from the published work (Paul et al., 2014 and NCI database compound ID NSC 32982). All the curcumin analogues showed higher sensitivity towards a panel of leukemia cell lines. A dose response curve of each compound (3b-g) against a panel of leukemia cell lines is given in Figure 3. The average sensitivity (MID) of all cancer cell lines towards the test agents (3a-g) was calculated in µM. The data showed that the MID for compound 3a was found to be 0.47 µM which was found to be comparatively less than that of the MID calculated for other curcumin analogues (Table 1). The order of sensitivity of compound 3a towards different panels of cell lines followed as leukemia, renal cancer, melanoma, non small cell lung cancer, prostate cancer, breast cancer, CNS cancer, colon cancer and ovarian cancer ( Figure 2). The dose response curve of compounds 3b-g is given in Figure 3 and the dose response curve of compounds 3a on the nine different panels of cell lines is given in Figure 4.

Discussion
Curcumin gained immense attention as a medicinal drug in modern medicine now a day. Undesirable side effect of synthetic pharmaceutical compelled us to search for natural approaches to disease prevention and treatment with the hope that naturally occurring compounds may be better tolerated than their synthetic counterparts. Hence we have taken curcumin as starting material and modified them to semi-synthetic analogues of curcumin as anticancer agent. All the compounds showed promising anticancer activity in single dose assay and met the predetermine criterion of growth inhibition and thus further chosen for the NCI full panel of five dose assay at five different drug concentrations (0.01, 0.1, 1, 10 and 100 µM). In five the dose assay all the curcumin analogues showed higher values of sensitivity towards the panel of leukemia cell lines. The average sensitivity (GI 50 ) of compound 3a to all cancer cell lines (NCI 60 cell lines) was found to be the highest among the tested compounds. The best result of TGI was observed on SR (leukemia) with 0.09 µM by compound 3a. All the curcumin analogues showed GI 50 between 0.03 µM (SR; leukemia) and 54.9 µM (SW620; colon cancer) and showed promising result in five dose assay. Some of the curcumin analogues reported earlier showed epidermal growth factor receptor (EGFR) tyrosine kinase as a potential target for anticancer activity (Ahsan et al., 2013). Furthermore we can say that the curcumin analogues evaluated here in five dose assay may perhaps targeted EGFR tyrosine kinase and showed anticancer activity. The anticancer activity of all these curcumin analogues are promising and hence could be subjected further for quantitative structure activity relationship (QSAR) studies to acquire more information and drug discovery.