Molecular mechanisms in C-Phycocyanin induced apoptosis in human chronic myeloid leukemia cell line-K562

Abstract

C-Phycocyanin (C-PC), the major light harvesting biliprotein from Spirulina platensis is of greater importance because of its various biological and pharmacological properties. It is a water soluble, non-toxic fluorescent protein pigment with potent anti-oxidant, anti-inflammatory and anti-cancer properties. In the present study the effect of highly purified C-PC was tested on growth and multiplication of human chronic myeloid leukemia cell line (K562). The results indicate significant decrease (49%) in the proliferation of K562 cells treated with 50 μM C-PC up to 48 h. Further studies involving fluorescence and electron microscope revealed characteristic apoptotic features like cell shrinkage, membrane blebbing and nuclear condensation. Agarose electrophoresis of genomic DNA of cells treated with C-PC showed fragmentation pattern typical for apoptotic cells. Flow cytometric analysis of cells treated with 25 and 50 μM C-PC for 48 h showed 14.11 and 20.93% cells in sub-G0/G1 phase, respectively. C-PC treatment of K562 cells also resulted in release of cytochrome c into the cytosol and poly(ADP) ribose polymerase (PARP) cleavage. These studies also showed down regulation of anti-apoptotic Bcl-2 but without any changes in pro-apoptotic Bax and thereby tilting the Bcl-2/Bax ratio towards apoptosis. These effects of C-PC appear to be mediated through entry of C-PC into the cytosol by an unknown mechanism. The present study thus demonstrates that C-PC induces apoptosis in K562 cells by cytochrome c release from mitochondria into the cytosol, PARP cleavage and down regulation of Bcl-2.

Introduction

Chemoprevention is an effective way to reduce cancer risk. Natural products have been the mainstay of cancer chemotherapy for the past 30 years. Blue-green algae are the most primitive life forms on earth with nutrient-dense, edible forms like Nostoc, Spirulina, Aphanizomenon species, etc. Spirulina is non-nitrogen fixing blue-green algae with over 30 years long history of safe human consumption. Spirulina is gaining attention as a nutraceutical and source of potential pharmaceuticals. Recent studies have demonstrated antioxidant [1], antimutagenic [2], antiviral [3], anticancer [4], [5], anti-allergic [6], immune enhancing [7], hepato-protective [8], blood vessel relaxing [9] and blood lipid-lowering effects [10] of Spirulina extracts. The biological and pharmacological properties of Spirulina were attributed mainly to calcium-spirulan and C-Phycocyanin (C-PC).

C-PC, a water-soluble non-toxic biliprotein pigment isolated from Spirulina platensis has significant antioxidant and radical scavenging properties [11]. Phycocyanin was shown to inhibit inflammation in mouse ears [12] and prevent acetic acid induced colitis in rats [13]. C-PC is used for the treatment of diseases such as Alzheimer’s and Parkinson’s [14] and prevents experimental oral and skin cancers [15]. Of major interest to ongoing research in inflammation as well as cancer is the finding that C-PC selectively inhibits cyclooxygenase-2 [16]. Recently we have reported that C-PC induces apoptosis in mouse macrophage cell line RAW 264.7 stimulated with LPS [17] and rat histiocytoma cell line AK5 [18]. C-PC caused a dose-dependent decrease in the levels of PGE₂ in LPS stimulated macrophage cell line [17]. This decrease in PGE₂ with no change in COX-1 and COX-2 protein levels could be due to inhibition of COX-2 activity by C-PC [16].

There is an expanding body of information on the potential applications of nonsteroidal anti-inflammatory drugs (NSAIDs) in cancer chemoprevention [19], [20]. Epidemiological studies indicate that the use of aspirin and other NSAIDs reduces the risk of cancer by 40–50% [21]. Selective inhibitors of COX-2 have been demonstrated to induce apoptosis in a variety of cancer cells, including those of colon [22], stomach [23], prostate and breast [24]. These observations are consistent with the cancer chemopreventive effects of NSAIDs. However, the biochemical mechanism underlying COX-2 inhibitor induced apoptosis remains elusive. Tumor inhibition by NSAIDs may be mediated by distinct cellular processes. These processes involve the ability of NSAIDs to restore apoptosis, induce cell cycle arrest, and inhibit angiogenesis [25], [26]. One of the main ways by which NSAIDs exert their effects is through modulation of apoptosis, although there is considerable debate about how these effects are mediated.

Since C-PC has many therapeutic roles including anticancer properties, the present study is undertaken to test the effect of highly purified (>95% pure) C-PC on growth and multiplication of human chronic myeloid leukemia K562 cell line and molecular mechanisms involved in C-PC induced cellular effects.