Molecular Study of Andrographolide on Cell Cycle, Apoptosis and Autophagy of Human Breast Carcinoma Cells

穿心蓮內酯對人類乳腺癌細胞的細胞週期、凋亡和自噬於分子平面上的研究

Student thesis: Doctoral Thesis

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Author(s)

  • Iyad A S ALQOUQA

Related Research Unit(s)

Detail(s)

Awarding Institution
Supervisors/Advisors
  • Hon Yeung CHEUNG (Supervisor)
Award date18 Jan 2017

Abstract

Breast cancer is the second most common cancer among women. It is a complex and heterogeneous disease with different biological characteristics and clinical behavior. Although many clinical and pathological features have been identified, management of breast cancer is still a challenging task due to lack of targeted therapy, notably in triple-negative breast cancer (TNBC), and the emergence of chemotherapy-resistant cell clones.

Recent studies have revealed that andrographolide (Andro) isolated from Andrographis paniculata Herba exhibited cytotoxic activity against a variety of human cancer cells. However, its effect on the proliferation, cell cycle, apoptosis and autophagy of human breast carcinoma cells has not been systematically investigated. In this study, two breast cancer cell lines, namely MCF7 and MDA-MB-231, in contrast to MCF10A, which is a noncancerous human mammary cell, were used as a model for elucidating the anticancer activities of Andro.

The cytotoxicity study of Andro showed a concentration and time-dependent growth inhibition on MCF7 and MDA-MB-231 with the IC50 of 29.0 μM and 24.7μM, respectively. Andro exhibited selective cytotoxicity towards the cancer cells without apparent toxicity on the normal cell, even at higher concentration (100μM).

Cell cycle analysis of Andro-treated cells revealed an arrest of G2/M in both MCF7 and MDA-MB-231 cells. During the G2/M arrest, alteration of the cell cycle regulatory proteins was noted to associate with upregulation of cyclin B1 and p21 Waf1/Cip and downregulation of P-cdc2 (Tyr15), cyclin D1, cyclin E, CDK 4 and CDK 6.

When the cells treated with Andro, some apoptotic features such as blebbing, chromatin condensation, DNA laddering were observed in MDA-MB-231. Meanwhile, blebbing or DNA laddering was absent in MCF7 cells. Nevertheless, cell death involved intrinsic mitochondrial pathway associated with the generation of reactive oxygen species (ROS), change in the mitochondrial membrane potential (MMP), and release of cytochrome-c from mitochondria.

Immunoblot analysis of Andro treated-MDA-MB-231 cells revealed cleaved caspase-3 and caspase-9 while the MCF7 cells lack caspase-3. Pretreatment of MDA-MB-231 with pan-caspase inhibitor, Z-VAD-FMK (20μM), partially protected the cells indicating the involvement of caspase-independent cell death (CICD). Translocation of mitochondrial AIF and EndoG implied CICD. Furthermore, pretreatment of the MDA-MB-231 cells with mitochondrial permeability transition pore (MPTP) inhibitor, Cyclosporine A (CsA, 1μM), could not rescue the cells from apoptosis.

Immunofluorescence and western analysis of DNA damage signaling revealed discrete foci of γH2AX in addition to upregulation of DNA damage and checkpoint markers p-Chk1 (S345), p-Chk2 (Thr68), p-P53 (S15) and p-BRCA1 (S1542). The BCl2 family proteins played a critical role in Andro induced apoptosis. In both cell lines, the antiapoptotic BCl2 was significantly downregulated in response to Andro treatment, whereas, proapoptotic proteins especially BAK, BIM and PUMA were upregulated. However, it was noticed that proapoptotic BAX upregulated in MCF7 while downregulated in MDA-MB-231 cells.

Andro induced early upstream autophagy, in both cell lines. Pretreatment of the cells with autophagy inhibitor 3MA (10mM), CQ (12μM), and BafA1 (200nM) augmented Ando-induced apoptosis indicating a pro-survival role of autophagy. Meanwhile, pretreatment with NAC (5mM), rescued the cells from apoptosis and autophagy showing apoptosis and autophagy are ROS-dependent. Immunoblot analysis in MDA-MB-231-treated cells, revealed that induction of autophagy occurred via inhibition of mTORC1 signaling pathway, and accompanied by upregulation of energy sensor AMPK. The results also suggested that autophagy, in MDA-MB-231, follows a non-canonical/non-classic pathway which is Beclin1 independent. The induction of autophagy in MCF7 developed through mTOR-independent mechanism accompanied by upregulation of p-ULK1 (S757), p-AKT (S473), and its downstream p-GSK3β(S9).

Western blot results further showed upregulation of ERK1/ERK2 in Andro treated cells, and the pharmacological inhibition of ERK using U0126 (20μM) or PD988059 (20μM) resulted in augmentation of apoptosis with the concomitant reduction in autophagy and amelioration of MMP collapse in both MCF7 and MDA-MB-231 cells. The inhibition of JNK in MDA-MB-231 cells using pharmacological inhibitor SP600125 (25μM) enhanced G2/M arrest and apoptosis and resulted in significant rise of ROS level. At the same time, SP600125 inhibited p-Akt (S473) and failed to block LC3B-II level. Finally, autophagy inducer, rapamycin, marginally diminished MMP collapse in Andro treated cells, while autophagy inhibitor 3MA, CQ significantly augmented apoptosis via increase of MMP loss.

In conclusion, the study shows that Andro induces apoptosis and protective autophagy via the ROS-dependent mechanism. It also reveals that cell death occurs through caspase-dependent and caspase-independent mechanisms, and, as a novel observation, the study indicates mTOR-dependent autophagy in MDA-MB-231 cells which is related to the beclin1-independent alternative pathway, while it demonstrates mTOR-independent autophagy in MCF7. This study further shows the inhibition of ERK in Ando-treated cells can lead to potentiation of apoptosis via increase of MMP and decrease of autophagy, whereas the JNK inhibition results in enhancement of apoptosis as well as LC3B-II level. Collectively, this study highlights the value and significance of Andro as a potential antineoplastic candidate for breast cancer and emphasizes the significant role of modulation of autophagy in the augmentation of the cytotoxic effect of Andro in breast cancer cells and provides valuable information towards understanding the mechanism of cell death and cell-type specific response.