Sreenivasa Rao Sagineedu

Phytochemicals from Phyllanthus niruri Linn. and their pharmacological properties: a review

This review discusses the medicinal plant Phyllanthus niruri Linn. (Euphorbiaceae), its wide variety of phytochemicals and their pharmacological properties. The active phytochemicals, flavonoids, alkaloids, terpenoids, lignans, polyphenols, tannins, coumarins and saponins, have been identified from various parts of P. niruri. Extracts of this herb have been proven to have therapeutic effects in many clinical studies. Some of the most intriguing therapeutic properties include anti‐hepatotoxic, anti‐lithic, anti‐hypertensive, anti‐HIV and anti‐hepatitis B. Therefore, studies relating to chemical characteristics and structural properties of the bioactive phytochemicals found in P. niruri are very useful for further research on this plant as many of the phytochemicals have shown preclinical therapeutic efficacies for a wide range of human diseases, including HIV/AIDS and hepatitis B.

Andrographolide and its analogues: versatile bioactive molecules for combating inflammation and cancer

1. Andrographis paniculata (Burm. f) Nees, commonly known as ‘king of bitters’, is a herbaceous plant belonging to the Family Acanthaceae. It has been widely used for centuries in Asian countries like China, India, Thailand and Malaysia for the treatment of sore throat, flu and upper respiratory tract infections.

Andrographolide derivatives inhibit guanine nucleotide exchange and abrogate oncogenic Ras function

Aberrant signaling by oncogenic mutant rat sarcoma (Ras) proteins occurs in ∼15% of all human tumors, yet direct inhibition of Ras by small molecules has remained elusive. Recently, several small-molecule ligands have been discovered that directly bind Ras and inhibit its function by interfering with exchange factor binding. However, it is unclear whether, or how, these ligands could lead to drugs that act against constitutively active oncogenic mutant Ras. Using a dynamics-based pocket identification scheme, ensemble docking, and innovative cell-based assays, here we show that andrographolide (AGP)—a bicyclic diterpenoid lactone isolated from Andrographis paniculata—and its benzylidene derivatives bind to transient pockets on Kirsten-Ras (K-Ras) and inhibit GDP–GTP exchange. As expected for inhibitors of exchange factor binding, AGP derivatives reduced GTP loading of wild-type K-Ras in response to acute EGF stimulation with a concomitant reduction in MAPK activation. Remarkably, however, prolonged treatment with AGP derivatives also reduced GTP loading of, and signal transmission by, oncogenic mutant K-RasG12V. In sum, the combined analysis of our computational and cell biology results show that AGP derivatives directly bind Ras, block GDP–GTP exchange, and inhibit both wild-type and oncogenic K-Ras signaling. Importantly, our findings not only show that nucleotide exchange factors are required for oncogenic Ras signaling but also demonstrate that inhibiting nucleotide exchange is a valid approach to abrogating the function of oncogenic mutant Ras.

NCI in vitro and in silico anticancer screen, cell cycle pertubation and apoptosis-inducing potential of new acylated, benzylidene and isopropylidene derivatives of andrographolide

Andrographolide (AGP) is the main bioactive constituent isolated from the traditional medicinal, Andrographis paniculata which contributes towards its various biological activities, including anticancer property. In this study, a series of new AGP derivatives were semi-synthesised and screened against the NCI in vitro 60 cell lines. From the screening results, we had identified SRS07 as the most potent AGP derivative, against breast and colon cancer cell lines. Subsequently, SRS07 was tested for its capability to induce cell cycle arrest and apoptosis in MCF-7 and HCT116 cancer cells. SRS07 effectively induced G1 cell cycle arrest in both cell lines and ultimately apoptosis by inducing DNA fragmentation in HCT116 cells. The apoptotic cell death induced by SRS07 was confirmed via FITC Annexin-V double staining. Western blot analysis of SRS07-treated HCT116 cells revealed that the compound induced apoptosis be activating caspase 8 which in turn cleaved Bid to t-Bid to initiate cell death cascade. Prediction of the possible mode of action of SRS07 by utilising NCI COMPARE analysis failed to reveal a distinct mechanism category. Hence, it is speculated that SRS07 possesses novel mechanism of action. In conclusion, SRS07 demonstrated superior in vitro anticancer profiles and emerged as a potential lead anticancer candidate.

A semisynthetic diterpenoid lactone inhibits NF-κB signalling to ameliorate inflammation and airway hyperresponsiveness in a mouse asthma model.

Andrographolide (AGP) and 14-deoxy-11,12-didehydroandrographolide (DDAG), two main diterpenoid constituents of Andrographis paniculata were previously shown to ameliorate asthmatic symptoms in a mouse model. However, due to inadequacies of both compounds in terms of drug-likeness, DDAG analogues were semisynthesised for assessment of their anti-asthma activity. A selected analogue, 3,19-diacetyl-14-deoxy-11,12-didehydroandrographolide (SRS27), was tested for inhibitory activity of NF-κB activation in TNF-α-induced A549 cells and was subsequently evaluated in a mouse model of ovalbumin (OVA)-induced asthma. Female BALB/c mice, 6-8weeks old were sensitized on days 0 and 14, and challenged on days 22, 23 and 24 with OVA. Compound or vehicle (3% dimethyl sulfoxide) was administered intraperitoneally 1h before and 11h after each OVA aerosol challenge. On day 25, pulmonary eosinophilia, airway hyperresponsiveness, mucus hypersecretion, inflammatory cytokines such as IL-4, -5 and -13 in BAL fluid, gene expression of inflammatory mediators such as 5-LOX, E-selectin, VCAM-1, CCL5, TNF-α, AMCase, Ym2, YKL-40, Muc5ac, CCL2 and iNOS in animal lung tissues, and serum IgE were determined. SRS27 at 30μM was found to suppress NF-κB nuclear translocation in A549 cells. In the ovalbumin-induced mouse asthma model, SRS27 at 3mg/kg displayed a substantial decrease in pulmonary eosinophilia, BAL fluid inflammatory cytokines level, serum IgE production, mucus hypersecretion and gene expression of inflammatory mediators in lung tissues. SRS27 is the first known DDAG analogue effective in ameliorating inflammation and airway hyperresponsiveness in the ovalbumin-induced mouse asthma model.

SRJ23, a new semisynthetic andrographolide derivative: in vitro growth inhibition and mechanisms of cell cycle arrest and apoptosis in prostate cancer cells.

Purpose3,19-(3-Chloro-4-fluorobenzylidene)andrographolide (SRJ23), a new semisynthetic derivative of andrographolide (AGP), exhibited selectivity against prostate cancer cells in the US National Cancer Institute (NCI) in vitro anti-cancer screen. Herein, we report the in vitro growth inhibition and mechanisms of cell cycle arrest and apoptosis induced by SRJ23.Methods3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used in assessing in vitro growth inhibition of compounds against prostate cancer (PC-3, DU-145 and LNCaP) and mouse macrophage (RAW 264.7) cell lines. Flow cytometry was utilised to analyse cell cycle distribution, whereas fluorescence microscopy was performed to determine morphological cell death. DNA fragmentation and annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry were done to confirm apoptosis induced by SRJ23. Quantitation of cell cycle and apoptotic regulatory proteins were determined by immunoblotting.ResultsAGP and SRJ23 selectively inhibited the growth of prostate cancer cells compared with RAW 264.7 cells at low micromolar concentrations; however, SRJ23 was more potent. Mechanistically, SRJ23-treated PC-3 cells displayed down-regulation of cyclin-dependent kinase (CDK) 1 without affecting levels of CDK4 and cyclin D1. However, SRJ23 induced down-regulation of CDK4 and cyclin D1 but without affecting CDK1 in DU145 and LNCaP cell lines. DNA histogram analysis revealed that the SRJ23 induced G2/M in PC-3 cells but G1 arrest in DU-145 and LNCaP cells. Morphologically, both compounds induced predominantly apoptosis, which was further confirmed by DNA fragmentation and annexin V-FITC staining. The DNA fragmentation was inhibited in the presence of caspase 8 inhibitor (Z-IETD-FMK). Apoptosis was associated with an increase in caspase 8 expression and activation. This thought to have induced cleavage of Bid into t-Bid. Additionally, increased expression and activation of caspase 9 and Bax proteins were apparent, with a concomitant down-regulation of Bcl-2 protein. Similar apoptosis cascade of events was observed in SRJ23-treated DU145 and LNCaP cell lines.ConclusionSRJ23 inhibited the growth of prostate cancer cells by inducing G2/M and G1 arrest via down-regulation of CDK1, and CDK4 and cyclin, respectively, and initiated caspase-8-mediated mitochondrial apoptosis. Taken together, these data support the potential of this compound as a new anti-prostate cancer agent.

SRJ09, a promising anticancer drug lead: Elucidation of mechanisms of antiproliferative and apoptogenic effects and assessment of in vivo antitumor efficacy

SRJ09 (3,19-(2-bromobenzylidene)andrographolide), a semisynthetic andrographolide (AGP) derivative, was shown to induce G1 cell cycle arrest and eventually apoptosis in breast and colon cancer cell lines. The present investigation was carried out to elucidate the mechanisms cell cycle arrest and apoptosis and evaluate the in vivo antitumor activity of SRJ09. The in vitro growth inhibitory properties of compounds were assessed in colon (HCT-116) and breast (MCF-7) cancer cell lines. Immunoblotting was utilized to quantitate the protein levels in cells. The gene expressions were determined using reverse transcriptase PCR (RT-PCR). Pharmacokinetic investigation was carried out by determining SRJ09 levels in plasma of Balb/C mice using HPLC. In vivo antitumor activity was evaluated in athymic mice carrying HCT-116 colon tumor xenografts. SRJ09 displayed improved in vitro activity when compared with AGP by producing rapid cell killing effect in vitro. Its activity was not compromised in MES-SA/Dx5 multidrug resistant (MDR) cells expressing p-glycoprotein. Cells treated with SRJ09 (0.1-10μM) displayed increased p21 protein level, which corresponded with gene expression. Whereas CDK4 protein level and gene expression was suppressed. The treatment did not affect cyclin D1. Changes of these proteins paralleled G1 cell cycle arrest in both cell lines as determined by flow cytometry. Induction of apoptosis by SRJ09 in HCT-116 cells which occurred independent of p53 and bcl-2 was inhibited in the presence of caspase 8 inhibitor, implicating the extrinsic apoptotic pathway. A single dose (100mg/kg, i.p) of SRJ09 produced a plasma concentration range of 12-30.4μM. At 400mg/kg (q4dX3), it significantly retarded growth of tumor xenografts. The antitumor activity of SRJ09 is suggested mediated via the induction of p21 expression and suppression of CDK-4 expression without affecting cyclin D1 to trigger G1 arrest leading to apoptosis.

Abstract 4454: SRJ09, a semisynthetic anticancer agent, targets Ras-MAPK signaling pathway: assessment in breast and colon cancer cell lines

SRJ09 (3,19-(2-bromobenzylidene)andrographolide) was shown to be more selective towards breast and colon cancers in USA NCI 60 cancer cell line screen, thus conferring it as lead anticancer agent (Jada et al (2008) Br J Pharmacol, 155(5):641-54). The compound was recently reported by us to be a mutant K-Ras binder (Hocker et al (2013), PNAS, 110(25):10201-6). In the present investigation, MCF-7 breast and HCT116 colon cancer cell lines were used to elucidate the mechanism of action of SRJ09. MTT cell viability assay was carried out to determine the growth inhibition by SRJ09 and other analogues of andrographolide (AGP). Cell cycle analysis was performed by flow cytometry technique. Western blot was carried out to determine the expression of cell signaling, cell cycle and apoptosis regulatory proteins. Additionally, reverse transcriptase (RT)-PCR was carried out to determine the expression of cell cycle regulatory genes. SRJ09 was approximately one-fold more potent than AGP and analogues. In terms of selectivity, HCT116 cells were more sensitive towards SRJ09 compared with MCF-7. SRJ09 was found to reduce the phosphorylated c-Raf and ERK1/2 proteins in both cell lines, with HCT116 being more susceptible towards the compound.SRJ09 induced G1 cell cycle arrest in both cell lines through up-regulation of p21 and down-regulation of CDK4 expressions, without affecting cyclin D1. However, HCT116 cells were more prone to undergo cell cycle arrest than MCF-7 cells. This finding was substantiated by western blot and RT-PCR analyses, whereby both the gene and protein expressions implicated in G1 arrest in HCT116 cells were affected at much lower concentration (3 µM) than in MCF-7. Western blot analysis revealed that SRJ09 induces apoptosis in both cell lines via p53-independent extrinsic pathway through activation of caspase 8 and with subsequent activation of Bax, Bid and caspase 9, without affecting Bcl-2. These findings were confirmed using caspase-8 inhibitor (Z-IETD-FMK), whereby HCT116 cells pretreated with the inhibitor failed to undergo apoptosis upon treatment with SRJ09. The differential sensitivity observed might be related to the K-Ras status in both cell lines, with HCT116 harboring mutant K-Ras while MCF-7 with wild type K-Ras. In conclusion, SRJ09 potentially inhibits K-Ras signaling pathway which reduces phosphorylated c-Raf and ERK1/2, thereby leading to G1 cell cycle arrest and apoptosis. Importantly, this study further supports our earlier finding that SRJ09 is a direct binder of K-Ras. This study was funded by Universiti Putra Malaysia Research University Grant Scheme (04-01-09-0713RU; 04-02-12-2017RU). Citation Format: Johnson Stanslas, Charng Choon Wong, Sreenivasa Rao Sagineedu, Shiran Mohd Sidik, Shariful Hasan Sumon, Roger Phillips, Nordin Haji Lajis. SRJ09, a semisynthetic anticancer agent, targets Ras-MAPK signaling pathway: assessment in breast and colon cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4454. doi:10.1158/1538-7445.AM2014-4454

Abstract C147: Mechanism of resistance to a semisynthetic anticancer andrographolide derivative: Possible involvement of Ras-MAPK signaling pathway.

SRJ09 (3,19-(2-bromobenzylidene)andrographolide) has been identified as a lead anticancer agent on the basis of its selectivity towards breast and colon cancers in the USA NCI 60 cancer cell lines screen (Jada et al (2008) Br J Pharmacol, 155(5):641-54). In our recent seminal publication (Hocker et al (2013), PNAS, 110(25):10201-6), we revealed that the compound is a binder of oncogenic mutant K-Ras. In the present investigation, HCT116 colon cancer cells were made resistant to SRJ09 to elucidate the mechanism of resistance. MTT cell viability assay was utilized to screen for cross-resistance to other analogues of andrographolide (AGP) and standard anticancer drugs. Flow cytometry technique was utilized for cell cycle study. Western blot was performed to determine the expression of cell cycle, apoptosis regulatory and cell signaling proteins. Additionally, reverse transcriptase (RT)-PCR was carried out to determine the cell cycle regulatory genes. SRJ09-resistant HCT116 displayed approximately 40-fold resistance towards SRJ09 compared with the parental cells. However, the cells were not resistant towards AGP analogues and standard anticancer drugs. The cells failed to arrest in G1 phase upon treatment with SRJ09, unlike the parental cells. RT-PCR confirmed the findings that protein expression of p21 was not up-regulated and CDK4 was not down-regulated, contrary to the parental cells. The resistance of the cells to undergo apoptosis upon treatment with SRJ09 was mainly attributed to the unchanged levels of Bax, Bid, caspase 8 and 9, otherwise activated in the parental cells. The inability of the cells to experience cell cycle arrest and apoptosis might be attributed in part to the marginal changes in phosphorylated c-Raf and ERK1/2 protein levels, which are in complete contrast to the parental cells. In conclusion, the mechanism of resistance is very likely due to failure of c-Raf and ERK1/2 to undergo dephosphorylation, implicating Ras-MAPK pathway as a target of SRJ09. This study was funded by Universiti Putra Malaysia Research University Grant Scheme (04-01-09-0713RU; 04-02-12-2017RU). Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C147. Citation Format: Johnson Stanslas, Charng Choon Wong, Sreenivasa Rao Sagineedu, Shiran Sidik, Shariful Hasan Sumon, Roger Phillips, Nordin Haji Lajis. Mechanism of resistance to a semisynthetic anticancer andrographolide derivative: Possible involvement of Ras-MAPK signaling pathway. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C147.