Thiol antioxidants sensitize malabaricone C induced cancer cell death via reprogramming redox sensitive p53 and NF-κB proteins in vitro and in vivo

Highlights

• Thiol antioxidants enhance sensitivity of lung cancer cells to mal C.

• Mal C induces generation of site-specific ROS and S-glutathionylation of redox sensitive proteins.

• P53 glutathionylation causes its higher accumulation in mitochondria, laeding to apoptosis.

• Mal C and thiol antioxidants effectively reduces lung tumor growth in mice.

Abstract

Specific focus on “redox cancer therapy” by targeting drugs to redox homeostasis of the cancer cells is growing rapidly. Recent clinical studies showed that N-acetyl cysteine (NAC) treatment significantly decreased the metabolic heterogeneity and reduced Ki67 (a proliferation marker) with simultaneous enhancement in apoptosis of tumor cells in patients. However, it is not yet precisely known how thiol antioxidants enhance killing of cancer cells in a context dependent manner. To this end, we showed that a dietary compound, malabaricone C (mal C) generated copious amounts of reactive oxygen species (ROS) and also reduced GSH level in lung cancer cells. Paradoxically, although antioxidants supplementation reduced mal C-induced ROS, thiol-antioxidants (NAC/GSH) restored intracellular GSH level but enhanced DNA DSBs and apoptotic cell death induced by mal C. Our results unraveled two tightly coupled biochemical mechanisms attributing this sensitization process by thiol antioxidants. Firstly, thiol antioxidants enable the “catechol-quinone redox cycle” of mal C and ameliorate ROS generation and bio-molecular damage (DNA and protein). Secondly, thiol antioxidants cause rapid glutathionylation of transcription factors [p53, p65 (NF-κB) etc.], oxidized by mal C, and abrogates their nuclear sequestration and transcription of the anti-apoptotic genes. Furthermore, analyses of the mitochondrial fractions of p53 expressing and silenced cells revealed that cytoplasmic accumulation of glutathionylated p53 (p53-SSG) triggers a robust mitochondrial death process. Interestingly, mutation of redox sensitive cysteine residues at 124, 141 and 182 position in p53 significantly reduces mal C plus NAC mediated sensitization of cancer cells. The preclinical results, in two different tumor models in mice, provides further support our conclusion that NAC is able to sensitize mal C induced suppression of tumor growth in vivo.

Introduction

Cancer is one of the leading causes of death in developing countries, and lung cancer contributes maximum cancer related mortality in men [1]. It contributes to 6.9% of new cases of cancers in India, and accounts for 19% of cancer related deaths worldwide [2]. Apart from primary lung cancers, lungs are the second most common sites for metastatic spread. In particular, the aggressive and highly metastatic late stage melanoma is well-known to induce secondary lung cancers [3]. The 5-year survival rate is less than 15% in patients with metastatic disease and approximately one-third of all melanoma patients experience disease recurrence [4]. Various chemotherapeutic agents are being exploited for management of lung cancer and metastasis. However, systemic hemato- and neuro-toxicity/side-effect and cancer recurrence due to inherent or acquired resistance remain major issues in cancer therapy [5]. Drugs that can modulate the actions of multiple targets may reduce/overcome the chances of drug resistance and recurrence in cancer therapy.

Several proteins like NF-κB, AP-1 (c-JUN/c-FOS), STAT3, c-MYC, p53 etc. play key roles in survival process of cancer cells and developing drug resistance [6]. Inhibition of the survival pathways and/or upregulation of the cell death process (by the JNK/p38 MAP-kinases etc) are believed to balance between these pathways to improve the chemotherapy outcome [7]. Use of pharmacological inhibitors to these survival proteins showed limited therapeutic applications due to their systemic toxicity. The constitutively up-regulated levels of reactive oxygen species (ROS), originating from metabolic alterations in malignant cells [8,9] provides an opportunity to target redox homeostasis in cancer cells. ROS modulate both anti-apoptotic, and pro-apoptotic effects to regulate tumorigenicity and malignant progression. Consistent with these, the antioxidants have been found to play dual role when administered with the anti-cancer drugs. For example, antioxidants (especially thiols) potentiated the efficacy of doxorubicin, leinamycin and gemcitabine against several human cancer cell lines and in murine xenografts [[10], [11], [12]]. But, presence of some antioxidants decreased cell death, induced by camptothecin, curcumin and other anti-cancer agents [13]. The dual roles of the antioxidants (especially the thiols) with the anti-neoplastic agents may be due to differential alteration in the ROS-induced cell death [14] and/or survival pathways [15,16]. Recently, S-glutathionylation of reactive cysteines, present in redox-sensitive proteins such as the metabolic enzymes, kinases, phosphatases, and transcription factors is emerging as a key post-translation modification. S-glutathionylation of various proteins alters the intracellular redox state and functional cellular responses [17,18]. Earlier, S-glutathionylation was found to inhibit NF-κB activity in hypoxia and gemcitabine-treated cells [12,14]. Elucidation of mechanism of protein glutathionylation, by altered redox homeostasis is expected to improve understanding of distinct cellular functions of these redox modified proteins. It may also provide the basis for novel approaches to treat cancer. Therefore, the glutathionylation-inducing agents that affect specific signaling routes, may be used as chemo-sensitizers to potentiate the efficacy of the cytotoxic drugs.

Considering the epidemiological evidence on the dietary influence on cancer prevention, extensive work is being pursued to identify natural compounds with the popular belief about their non-toxicity to humans [19]. Also, 50% of the internationally approved chemotherapeutic drugs are derived from natural products [20]. Spices are present in most of our daily diets, and consumption of spice rich food has been found to play an important role in suppressing the transformative, hyper-proliferative and inflammatory processes that initiate carcinogenesis [21]. Previously we have shown that malabaricone C (mal C, Fig. 1A), a phenolic constituent of an Indian spice, Myristica malabarica possesses superior in vitro antioxidant activity than curcumin [22], but causes ROS-mediated DNA damage in presence of Cu(II) [23] and lysosomal membrane permeabilization in cancer cells [24]. Mal C was also found to induce DNA damage in human lung cancer cells by activating ATM/CHK1-p38-dependent mitochondrial membrane disruption that led to caspase-dependent cell death [25]. Although mal C produced significant amount of ROS in the lung cancer cells [25], but its consequence on mal C induced cytotoxicity remained unresolved. Hence, the primary aim of this study was to assess the importance of ROS in mal C-induced lung cancer cell death and its modulation by antioxidants. Our results revealed that the thiol antioxidants (NAC/GSH) sensitize the lung cancers towards mal C. The mal C and NAC/GSH combinations induced S-glutathionylation (a reversible modification) of proteins like p65 (NF-κB) and p53, thus sequestering them in the cytoplasm and abrogating their transcriptional role to induce mitochondrial death process.