3β-Hydroxylup-20(29)-ene-27,28-dioic acid dimethyl ester, a novel natural product from Plumbago zeylanica inhibits the proliferation and migration of MDA-MB-231 cells

Abstract

Plumbago zeylanica, a traditional Indian herb is being used for the therapy of rheumatism and has been approved for anti-tumor activity. However, the molecular mechanisms involved in the biological action are not very well understood. In this study, the anti-invasive activities of P. zeylanica methanolic extract (PME) and pure compound 3β-hydroxylup-20(29)-ene-27,28-dioic acid (PZP) isolated from it are investigated in vitro. PME and PZP were noted to have the ability to induce apoptosis as assessed by flow cytometry. Further, the molecular mechanism of apoptosis induced by PME and PZP was found by the loss of mitochondrial membrane potential with the down regulation of Bcl-2, increased expression of Bad, release of cytochrome c, activation of caspase-3 and cleavage of PARP leading to DNA fragmentation. Importantly, both PME and PZP were observed to suppress MDA-MB-231 cells adhesion to the fibronectin-coated substrate and also inhibited the wound healing migration and invasion of MDA-MB-231 cells through the reconstituted extracellular matrix. Gelatin zymography revealed that PME and PZP decreased the secretion of matrix metalloproteinases-2 (MMP-2) and metalloproteinases-9 (MMP-9). Interestingly both PME and PZP exerted an inhibitory effect on the protein levels of p-PI3K, p-Akt, p-JNK, p-ERK1/2, MMP-2, MMP-9, VEGF and HIF-1α that are consistent with the observed anti-metastatic effect. Collectively, these data provide the molecular basis of the anti-proliferative and anti-metastatic effects of PME and PZP.

Introduction

Breast cancer, one of the most common cancers in the world, accounts for approximately 20% mortality and is capable of metastasizing to local lymph nodes as well as to distal organs such as bone, lung and liver [1]. About 76% of all breast tumors have been categorized as invasive breast cancers [2]. Currently available anti-cancer drugs induce apoptosis either via mitochondrial (intrinsic) pathway which is initiated by multiple sensors to induce DNA damage, mitochondrial outer membrane permeabilization (MOMP) and release of cytochrome c from the mitochondria into cytosol [3] or the death receptor (extrinsic) pathway that is stimulated by cell surface death receptors such as tumor necrosis factor (TNF) receptor and Fas. In the presence of an apoptotic stimulus, Bad is dephosphorylated translocating to the mitochondria where it binds with Bcl-2 to exert its pro-apoptotic activity [4]. In both the intrinsic and the extrinsic pathways, the activated caspases-9 and -8 have been observed to play a significant role in the activation of caspase-3 [5], [6].

Metastasis is a complex and multi-step dispersion process of malignant tumor cells from the primary tumor site to a secondary site within the body [7]. Therefore, activation of zinc-binding endopeptidases MMPs [(MMP-2) and MMP-9)] is considered to play a crucial role in the process of cancer invasion and metastasis. MMPs are primarily regulated at the transcriptional (through AP-1 or NF-κB via mitogen activated protein kinase (MAPK) or PI3K-Akt pathways) level, at posttranscriptional levels, at the protein level via their activators or inhibitors, or at their cell surface localization [8]. Over expression of MMP-2 and MMP-9 in malignant tumors has been observed to develop vasculature by angiogenesis. The main factor eliciting angiogenesis is hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) [9].

In the global scenario the lowest prevalence of breast cancer is among Asian women [10] because of their high consumption of dietary plants such as Soy, Turmeric, etc., [11]. The present study investigates Plumbago zeylanica (Family: Plumbaginaceae), a common household plant remedy for diarrhea in India [12], also reported for the treatment of cancer, malaria, rheumatism, dyspepsia and piles [13], [14]. The major constituents in P. zeylanica are naphthoquinones, steroids, sugars, naphthalenones, alkanes, triterpenes and amino acids [15]. Plumbagin, a napthoquinone analogue from P. zeylanica, has been extensively studied for the inhibition of proliferation of variety of cell lines and animal models [16].

We have investigated the small molecule isolated from the crude extract of P. zeylanica by bioactivity guided fractionation using MDA-MB-231 cells for its anti-proliferative activity and its mechanism of apoptosis and metastasis was elucidated by studying the key markers involved. In this study, the effects of P. zeylanica methanolic extract (PME) and pure compound 3β-hydroxylup-20(29)-ene-27, 28-dioic acid (PZP), a pentacyclic triterpene, on p-PI3K, p-Akt, Bcl-2, Bad, cytochrome c, caspase-3, PARP as well as p-JNK, p-ERK1/2, MMPs, VEGF and HIF-1α expression were examined on MDA-MB-231 cells, a highly metastatic human breast carcinoma cells, to explore the underlying mechanism for the involvement of PME and PZP in cancer cell proliferation and invasion.