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Andrographolide (Andro) has emerged recently as a potential and effective anticancer agent with induction of apoptosis in some cancer cell lines while induction of G2/M arrest with weak apoptosis in others. Few studies have proved that Andro is also effective in combination therapy. The flavonoid Taxifolin (Taxi) has showed anti-oxidant and antiproliferative effects against different cancer cells. Therefore, the present study investigated the cytotoxic effects of Andro alone or in combination with Taxi on HeLa cells. The combination of Andro with Taxi was synergistic at all tested concentrations and combination ratios. Andro alone induced caspase-dependent apoptosis which was enhanced by the combination with Taxi and attenuated partly by using Z-Vad-Fmk. Andro induced a protective reactive oxygen species (ROS)-dependent autophagy which was attenuated by Taxi. The activation of p53 was involved in Andro-induced autophagy where the use of Taxi or pifithrin-α (PFT-α) decreased it while the activation of JNK was involved in the cell death of HeLa cells but not in the induction of autophagy. The mitochondrial outer-membrane permeabilization (MOMP) plays an important role in Andro-induced cell death in HeLa cells. Andro alone increased the MOMP which was further increased in the case of combination. This led to the increase in AIF and cytochrome c release from mitochondria which consequently increased caspase-dependent and independent cell death. In conclusion, Andro induced a protective autophagy in HeLa cells which was reduced by Taxi and the cell death was increased by increasing the MOMP and subsequently the caspase-dependent and independent cell death., Competing Interests: The authors have declared that no competing interests exist.

Andrographolide (Andro) suppresses proliferation and triggers apoptosis in many types of cancer cells. Taxifolin (Taxi) has been proposed to prevent cancer development similar to other dietary flavonoids. In the present study, the cytotoxic and apoptotic effects of the addition of Andro alone and Andro and Taxi together on human prostate carcinoma DU145 cells were assessed. Andro inhibited prostate cancer cell proliferation by mitotic arrest and activation of the intrinsic apoptotic pathway. Although the effect of Taxi alone on DU145 cell proliferation was not significant, the combined use of Taxi with Andro significantly potentiated the anti-proliferative effect of increased mitotic arrest and apoptosis by enhancing the cleavage of poly(ADP-ribose) polymerase, and caspases-7 and -9. Andro together with Taxi enhanced microtubule polymerization in vitro, and they induced the formation of twisted and elongated spindles in the cancer cells, thus leading to mitotic arrest. In addition, we showed that depletion of MAD2, a component in the spindle assembly checkpoint (SAC), alleviated the mitotic block induced by the two compounds, suggesting that they trigger mitotic arrest by SAC activation. This study suggests that the anti-cancer activity of Andro can be significantly enhanced in combination with Taxi by disrupting microtubule dynamics and activating the SAC.

Due to the poor water solubility of andrographolide (andro), an inclusion technique has been developed to modify its physical and chemical properties so as to improve its bioavailability. In contrast with the immense experimental studies on the inclusion complexes of andro:cyclodextrin, no computational study has so far been carried out on this system. In this work, preliminary docking experiments with AutoDock were performed. Density Functional Theory (DFT) and Austin Model 1 (AM1) calculations upon the docking instances were applied to investigate the two possible modes of molecular inclusions between andro and x-cyclodextrin (xCD, where x is alpha, beta or gamma). Atoms-in-Molecules (AIM) analysis based on the B3LYP/cc-pVDZ wavefunction was applied to verify the existence of the intermolecular hydrogen bonds. It was found that the most stable complex among the six possible inclusion complexes was the one formed between andro and betaCD with andro's decalin ring moiety wrapped by CD at a ratio of 1:1. The hydrogen bonds between andro and CD were responsible for the stability of the inclusion complexes. The calculated data were found to be consistent with the experimental results. Thus, the results of this study can aid new drug design processes.

The intracellular level of glutathione (GSH) was significantly decreased after the addition of andrographolide (1) to cell cultures of HepG2. When the molecular interaction between andrographolide and GSH was investigated under a condition mimicking the in vivo environment, we observed that the level of GSH dropped in the presence of andrographolide. Stoichiometric analysis indicates that the reaction between these two reactants was 1 to 1 at pH 7 and followed second order kinetics. The activation energy of the overall reaction was 41.9+/-10 kJ x mol(-1) according to the Arrhenius equation. Using a micro-liquid-liquid extraction method followed by micellar electrokinetic chromatographic separation, two major products were isolated and identified, and their chemical structures were determined as 14-deoxy-12-(glutathione-amino)-andrographolide (2) and 14-deoxy-12-(glutathione-S-yl)-andrographolide (3). Based on these structural findings, a hypothetical mechanism of reaction between glutathione and andrographolide was proposed. It is concluded that the alpha,beta-unsaturated lactone moiety of andrographolide reacts with GSH through a Michael addition followed by dehydration of the adduct.

The cytotoxicity of andrographolide to HepG2 human hepatoma cells was investigated in the present study. Growth of HepG2 cells was affected in the presence of andrographolide with an IC(50) of 40.2 microM after 48 h treatment. Flow cytometric analysis and DNA fragmentation assay revealed that andrographolide induced cell cycle arrest at G2/M phase and a late apoptosis of the cells. The occurrence of cell cycle arrest was accompanied by the collapse of mitochondrial membrane potential (MMP) and an intracellular increase of hydrogen peroxide (H(2)O(2)) but a decrease of superoxide radicals (O(2)(-)) and reduced glutathione. In the treated cells, expression of Bax as well as the transcriptional controller of this pro-apoptotic gene, p53, was upregulated but not other apoptotic proteins such as Bad, Bcl-2 and Bcl-X(L). Although the activity of caspase-3, which has direct effect on apoptosis, was also enhanced by the presence of andrographolide, cell death of HepG2 could neither be prevented by a specific inhibitor of capsase-3 nor the pan-caspase inhibitor-zVAD (Val-Ala-Asp), indicating that it was a caspase-independent cell death. Since the overall percentage of apoptotic cells was relatively small throughout the experimental studies, we conclude that the cytotoxic effect of andrographolide on HepG2 cells is primary attributed to the induction of cell cycle arrest via the alteration of cellular redox status.

Triptolide, a bioactive component of the Chinese medicinal herb Tripterygium wilfordii Hook F., induces p53-mediated apoptosis in cancer cells. This study demonstrated that triptolide activated an alternative p53-independent apoptotic pathway in HL-60 cells. In the absence of an intact p53 and without changing Bax level, at nM range triptolide induced apoptosis with concomitant DNA fragmentation, S phase cell cycle arrest, mitochondrial cytochrome c release and the activation of caspases. Besides, both caspases 8 and 9 were activated and the simultaneous inhibition of both was required to completely block triptolide's apoptotic effect. Importantly, triptolide induced the appearance of a truncated 23kD Bcl-2 which was inhibited by the general caspase inhibitor Z-VAD-FMK. In the MCF-7 cells that possessed the wild type p53 but lacked caspases 3, triptolide induced cell death with an increase in p53 but Bcl-2 remained unaltered. On the other hand, transfected cells overexpressing the 28kD Bcl-2 became more resistant to triptolide and upon triptolide treatment accumulated in the G(1) instead of S phase. After 36h treatment, triptolide activated JNK pathways, at the same time inactivated the ERK and p38 pathways. However, SP600125, a specific JNK inhibitor, could not inhibit the triptolide-mediated cleavage of caspase 3, indicated that activation of JNK might not be related to the apoptotic effects of triptolide. Our data suggest that in the absence of an intact p53 and without altering Bax level triptolide induces apoptosis activates a positive amplification loop involving caspase-mediated Bcl-2 cleavage/activation, mitochondrial cytochrome c release and further activation of caspases.

The in vitro cytotoxicities of the ethanol extract of Andrographis paniculata (APE) and its main diterpenoid components were evaluated in various cancer cells. APE was found to be significantly growth inhibitory to human acute myeloid leukemic HL-60 cells with an IC (50) value of 14.01 microg/mL after 24 h of treatment. Among the three main diterpenoids in A. paniculata, andrographolide exhibited the highest degree of cytotoxicity followed by deoxyandrographolide while neoandrographolide was the least effective. Laser confocal microscopy and gel electrophoresis studies revealed chromosomal DNA fragmentations suggesting the occurrence of apoptosis. An increase of G (0)/G (1) phase cells from 51.88 % to 78.69 % was noted after andrographolide treatment for 36 h. The G (0)/G (1) phase arrest and apoptosis were associated with disappearance of mitochondrial cytochrome c and increased expression of Bax but decreased expression of Bcl-2 proteins in the inhibited cells. Although the order of all these events has not been determined, it is concluded that APE and andrographolide induce cell cycle arrest and affect an intrinsic mitochondria-dependent pathway of apoptosis by regulating the expression of some pro-apoptotic markers in HL-60 cells.