Ilora Ghosh

Excessive reactive oxygen species induces apoptosis in fibroblasts : Role of mitochondrially accumulated hyaluronic acid binding protein 1 (HABP1/p32/gC1qR)

Constitutively expressed HABP1 in normal murine fibroblast cell line induces growth perturbation, morphological abnormalities along with initiation of apoptosis. Here, we demonstrate that though HABP1 accumulation started in mitochondria from 48 hr of growth, induction of apoptosis with the release of cytochrome c and apoptosome complex formation occurred only after 60 hr. This mitochondrial dysfunction was due to gradual increase in ROS generation in HABP1 overexpressing cells. Along with ROS generation, increased Ca 2+ influx in mitochondria leading to drop in membrane potential was evident. Interestingly, upon expression of HABP1, the respiratory chain complex I was shown to be significantly inhibited. Electronmicrograph confirmed defective mitochondrial ultrastructure. The reduction in oxidant generation and drop in apoptotic cell population accomplished by disruption of HABP1 expression, corroborating the fact that excess ROS generation in HABP1 overexpressing cells leading to apoptosis was due to mitochondrial HABP1 accumulation.

Hyaluronan-binding protein 1 (HABP1/p32/gC1qR) induces melanoma cell migration and tumor growth by NF-kappa B dependent MMP-2 activation through integrin αvβ3 interaction

Cell migration is the hallmark of cancer regulating anchorage independent growth and invasiveness of tumor cells. Hyaluronan (HA), an ECM polysaccharide is shown to regulate this process. In the present report, we demonstrated, supplementation of purified recombinant hyaluronan binding protein 1(HABP1/p32/gC1qR) from human fibroblast cDNA enhanced migration potential of highly invasive melanoma (B16F10) cells. Exogenous HABP1 adhered to the cell surface transiently and was shown to interact and colocalize with α(v)β(3) integrin, a regulatory molecule of cell migration. In HABP1 treated cells, the phosphorylation of nuclear factor inducing kinase (NIK) and IκBα was observed, followed by nuclear translocation of p65 subunit of NFκB, along with its DNA-binding and transactivation, resulting in upregulation of MT1-MMP expression and finally MMP-2 activation. To substantiate our findings, prior to HABP1 treatment, the expression of NIK was reduced by small interfering RNA mediated knockdown and confirmed the inhibition of nuclear translocation of p65 subunit of NFκB and upregulation of MT1-MMP expression. In addition, the use of curcumin, an anti-cancer drug, or GRGDSP, the blocking peptide along with exogenous HABP1, inhibited such NFκB-dependent pathway, confirming that HABP1-induced cell migration is α(v)β(3) integrin-mediated and downstream signaling by NFκB. Finally, we translated the in vitro data in mice model and observed enhanced tumor growth with higher MT1-MMP expression and MMP-2 activation in the tumors upon injection of HABP1 treated melanoma cells. The treatment of curcumin, the anticancer drug along with HABP1, inhibited the migration, expression of MT1-MMP and activation of MMP-2 and finally tumor growth supports the involvement of HABP1 in tumor formation.

Differential expression of Hyaluronic Acid Binding protein 1 (HABP1)/P32/C1QBP during progression of epidermal carcinoma

Our laboratory has characterized a novel cell surface glycoprotein, Hyaluronic Acid Binding Protein 1 (HABP1), interacting specifically with hyaluronan (HA) and regulating HA-mediated cellular event. The involvement of HA in different stages of carcinoma is well documented. In the present communication, the expression profile of HABP1 was investigated from initiation to progression of epidermal carcinoma in mice, induced by benzo[a]pyrene (B[a]P) exposure. During tumor initiation, HABP1 accumulated in inflammatory subsquamous tissue and with progression, the protein, was also seen to overexpress in papillomatic and acanthotic tissue. With the onset of metastasis, HABP1 overexpression was confined to metastatic islands, while it disappeared gradually from the surrounding mass. Such expression profiles in metastasized tissue were supported by decreased levels of HABP1, both at protein and transcript levels. These observations taken together suggest that the changes in HABP1 level coincide with specific stages of tumor progression, that lead to disruption of its interaction with HA, implying a role in the regulation of tumor metastasis. (Mol Cell Biochem 267: 133–139, 2004)

Reduction in the level of hyaluronan binding protein 1 (HABP1) is associated with loss of sperm motility

Hyaluronan binding protein 1 (HABP1) was reported to be present on human sperm surface and its involvement in fertilization has already been elucidated (Mol. Repro. Dev. 38 (1994) 69). In the present communication, we report a significant reduction in the level of this protein in sperms from asthenozoospermic and oligozoospermic patients as compared to normozoospermic one. Further evidence of the absence of HABP1 in sperms, having motility <20% is documented, which again is a determining factor for fertilization. HABP1 was quantitatively determined using anti-HABP1 antibody from sperm extracts isolated from semen samples of both the fertile and infertile groups demonstrating low sperm motility. Sperm samples with low motility revealed a significant reduction in the level of HABP1 in immunoblot detection as well as immunolocalization experiment. It suggests that decreased HABP1 level may be associated with low motility of sperms, which in turn might cause infertility in the patient. Thus, the sperm surface HABP1 level can be correlated with the degree of sperm motility, an important criteria for fertilization.

Overexpression of Hyaluronan-binding Protein 1 (HABP1/p32/gC1qR) in HepG2 Cells Leads to Increased Hyaluronan Synthesis and Cell Proliferation by Up-regulation of Cyclin D1 in AKT-dependent Pathway

Background: Hyaluronan (HA) levels regulate cell behavior, tumor invasion, and migration through interactions with hyaladherins. Results: Elevated expression of hyaluronan-binding protein 1 (HABP1) leads to enhanced HA synthesis, HA cable formation, and activation of cell survival pathways in HepG2 cells. Conclusion: Constitutively elevated expression of HABP1 leads to enhanced tumorigenic potential by HA-mediated pathways. Significance: HABP1 modulates cell survival through enhanced HA synthesis. Overexpression of the mature form of hyaluronan-binding protein 1 (HABP1/gC1qR/p32), a ubiquitous multifunctional protein involved in cellular signaling, in normal murine fibroblast cells leads to enhanced generation of reactive oxygen species (ROS), mitochondrial dysfunction, and ultimately apoptosis with the release of cytochrome c. In the present study, human liver cancer cell line HepG2, having high intracellular antioxidant levels was chosen for stable overexpression of HABP1. The stable transformant of HepG2, overexpressing HABP1 does not lead to ROS generation, cellular stress, and apoptosis, rather it induced enhanced cell growth and proliferation over longer periods. Phenotypic changes in the stable transformant were associated with the increased “HA pool,” formation of the “HA cable” structure, up-regulation of HA synthase-2, and CD44, a receptor for HA. Enhanced cell survival was further supported by activation of MAP kinase and AKT-mediated cell survival pathways, which leads to an increase in CYCLIN D1 promoter activity. Compared with its parent counterpart HepG2, the stable transformant showed enhanced tumorigenicity as evident by its sustained growth in low serum conditions, formation of the HA cable structure, increased anchorage-independent growth, and cell-cell adhesion. This study suggests that overexpression of HABP1 in HepG2 cells leads to enhanced cell survival and tumorigenicity by activating HA-mediated cell survival pathways.

A Combined Approach against Tumorigenesis using Glucose Deprivation and Mitochondrial Complex 1 Inhibition by Rotenone

Cancer cells exhibit various degrees of mitochondrial metabolic alterations. Owing to their multiple roles, mitochondria are attractive target for cancer therapy. Cancerous cells have high glucose (HG) requirements for their growth. Depriving them of glucose has been an approach used in many studies to restrict their perpetuation. However, such deprivation can negatively affect the surrounding normal cells in vivo. Keeping this in view, we treated HeLa cells with only physiological glucose (PG, 5.5 mM) and a combination of physiological glucose with a very low dose (1 nM) of rotenone (PGT), taking high glucose (HG, 25 mM)‐treated HeLa cells as normal. We demonstrated that HeLa cells under PG condition mainly exhibited growth arrest. The PGT combination induced apoptosis in HeLa cells by generation of ROS, decrease in ATP production even with around 1.89‐fold increase in glucose consumption, cell cycle arrest at S‐phase and substantial increase in sub‐diploid (Sub‐D) population. The oxidative stress generated in both PG and PGT conditions stabilised p53 by localising it in the nuclei of HeLa cells, which would have otherwise undergone HPV‐mediated inactivation. Pre‐mature senescence induced due to limited glucose availability was found to be regulated by nuclear translocated p53 which, in turn, induced p21, pAkt and pERK. The cyto‐toxic effect of rotenone on glucose deprived HeLa cells, synergistically activated NFκB, caspase‐3 and Bax along with reduced expression of hyaluronan, a ROS scavenging molecule on their cell surface. Thus, our finding might be a valuable approach to specifically target cancerous cells in a more physiologically feasible condition and can serve as a relevant biochemical basis to gain new insights into cancer therapy.

Evidence for Serpentine as a novel antioxidant by a redox sensitive HABP1 overexpressing cell line by inhibiting its nuclear translocation of NF-κB

Abstract Herbal antioxidants are gradually gaining importance as dietary supplements considering the growing implications of oxidative stress in most degenerative diseases and aging. Thus, continuous attempts are made to search for novel herbal molecules with antioxidative properties, using chemical methods predominantly with the need arising for cell based assays. We have generated a stable cell line F-HABP07, by constitutively overexpressing human Hyaluronan Binding Protein1 (HABP1) in murine fibroblasts which accumulates in the mitochondria leading to excess ROS generation without any external stimuli. In the present study, we demonstrated the nuclear translocation of p65 subunit of NF-κB in F-HABP07 cells, an important signature of ROS induced signalling cascade providing us an opportunity to use it as a screening system for ROS scavengers. Using known antioxidants on our designer cell line, we have demonstrated a dose dependant reduction in ROS generation and observed inhibition of p65 subunit of NF-κB nuclear translocation, increase in glutathione content and down-regulation of apoptotic marker Bax establishing its antioxidant biosensing capacity. With the help of this cell line, we for the first time demonstrated serpentine, one of the active components from the roots of Rauwolfia serpentina (a traditional medicinal plant), to be a novel non-cytotoxic antioxidant. The authenticity of this cell line screening system based discovery was validated using standard chemical assays thus, opening up new therapeutic avenues for this herbal compound and the use of this designer cell line.

Brain most susceptible to cadmium induced oxidative stress in mice.

Accumulated evidence over the years indicate that cadmium (Cd) may be a possible etiological factor for neurodegenerative diseases. This may possibly be linked to excessive generation of free radicals that damages the organs in the body depending on their defence mechanism. Since Cd is a toxic agent that affect several cell types, the aim of this study was to shed light on the effect of Cd and its consequences on different organs of the mice body. To test the hypothesis of concentration dependent Reactive Oxygen Species (ROS) generation and DNA damage, observations were done in the serum of 4-5 weeks old male Swiss albino mice by treating with cadmium chloride (CdCl2) in drinking water for 30 days. The expression of Bcl-2-associated X protein (Bax) an apoptotic marker protein was two times higher in brain compared to liver at an exposure level of 0.5mgL(-1) CdCl2. Furthermore the correlation and linkage data analysis of antioxidant defence system revealed a rapid alteration in the brain, compared to any other organs considered in this study. We report that even at low dose of Cd, it impaired the brain due to lipid peroxidase sensitivity which favoured the Cd-induced oxidative injury in the brain.

Autophagic Vacuolation Induced by Excess ROS Generation in HABP1/p32/gC1qR Overexpressing Fibroblasts and Its Reversal by Polymeric Hyaluronan

The ubiquitous hyaladherin, hyaluronan-binding protein 1 (HABP1/p32/gC1qR) upon stable overexpression in normal fibroblasts (F-HABP07) has been reported to induce mitochondrial dysfunction, growth retardation and apoptosis after 72 h of growth. HABP1 has been observed to accumulate in the mitochondria resulting in generation of excess Reactive Oxygen Species (ROS), mitochondrial Ca++ efflux and drop in mitochondrial membrane potential. In the present study, autophagic vacuolation was detected with monodansylcadaverin (MDC) staining from 36 h to 60 h of culture period along with elevated level of ROS in F-HABP07 cells. Increased expression of autophagic markers like MAP-LC3-II, Beclin 1 and autophagic modulator, DRAM confirmed the occurrence of the phenomenon. Reduced vacuole formation was observed upon treatment with 3-MA, a known PI3 kinase inhibitor, only at 32 h and was ineffective if treated later, as high ROS level was already attained. Treatment of F111 and F-HABP07 cells with bafilomycin A1 further indicated an increase in autophagosome formation along with autophagic degradation in HABP1 overexpressed fibroblasts. Comparison between normal fibroblast (F111) and F-HABP07 cells indicate reduced level of polymeric HA, its depolymerization and perturbed HA-HABP1 interaction in F-HABP07. Interestingly, supplementation of polymeric HA, an endogenous ROS scavenger, in the culture medium prompted reduction in number of vacuoles in F-HABP07 along with drop in ROS level, implying that excess ROS generation triggers initiation of autophagic vacuole formation prior to apoptosis due to overexpression of HABP1. Thus, the phenomenon of autophagy takes place prior to apoptosis induction in the HABP1 overexpressing cell line, F-HABP07.

Increased Hyaluronan Levels in HABP1/p32/gC1qR Overexpressing HepG2 Cells Inhibit Autophagic Vacuolation Regulating Tumor Potency

Tumor growth and development is influenced by its microenvironment. A major extracellular matrix molecule involved in cancer progression is hyaluronan (HA). Hyaluronan and expression of a number of hyaladherin family proteins are dramatically increased in many cancer malignancies. One such hyaladherin, hyaluronan-binding protein 1 (HABP1/p32/gC1qR) has been considered to be a biomarker for tumor progression. Interestingly, overexpression of HABP1 in fibroblast has been shown to increase autophagy via generation of excess reactive oxygen species (ROS) and depletion of HA leading to apoptosis. Cancerous cells are often found to exhibit decreased rate of proteolysis/autophagy in comparison to their normal counterparts. To determine if HABP1 levels alter tumorigenicity of cancerous cells, HepR21, the stable transfectant overexpressing HABP1 in HepG2 cell line was derived. HepR21 has been shown to have increased proliferation rate than HepG2, intracellular HA cable formation and enhanced tumor potency without any significant alteration of intracellular ROS. In this paper we have observed that HepR21 cells containing higher endogenous HA levels, have downregulated expression of the autophagic marker, MAP-LC3, consistent with unaltered levels of endogenous ROS. In fact, HepR21 cells seem to have significant resistance to exogenous ROS stimuli and glutathione depletion. HepR21 cells were also found to be more resilient to nutrient starvation in comparison to its parent cell line. Decline in intracellular HA levels and HA cables in HepR21 cells upon treatment with HAS inhibitor (4-MU), induced a surge in ROS levels leading to increased expression of MAP-LC3 and tumor suppressors Beclin 1 and PTEN. This suggests the importance of HABP1 induced HA cable formation in enhancing tumor potency by maintaining the oxidant levels and subsequent autophagic vacuolation.