Ipsita Pal

PI3K and Akt as molecular targets for cancer therapy: current clinical outcomes

The PI3K-Akt pathway is a vital regulator of cell proliferation and survival. Alterations in the PIK3CA gene that lead to enhanced PI3K kinase activity have been reported in many human cancer types, including cancers of the colon, breast, brain, liver, stomach and lung. Deregulation of PI3K causes aberrant Akt activity. Therefore targeting this pathway could have implications for cancer treatment. The first generation PI3K-Akt inhibitors were proven to be highly effective with a low IC50, but later, they were shown to have toxic side effects and poor pharmacological properties and selectivity. Thus, these inhibitors were only effective in preclinical models. However, derivatives of these first generation inhibitors are much more selective and are quite effective in targeting the PI3K-Akt pathway, either alone or in combination. These second-generation inhibitors are essentially a specific chemical moiety that helps to form a strong hydrogen bond interaction with the PI3K/Akt molecule. The goal of this review is to delineate the current efforts that have been undertaken to inhibit the various components of the PI3K and Akt pathway in different types of cancer both in vitro and in vivo. Our focus here is on these novel therapies and their inhibitory effects that depend upon their chemical nature, as well as their development towards clinical trials.

Antineoplastic and Apoptotic Potential of Traditional Medicines Thymoquinone and Diosgenin in Squamous Cell Carcinoma

Thymoquinone (TQ) and diosgenin (DG), the active ingredients obtained from black cumin (Nigella sativa) and fenugreek (Trigonella foenum graecum), respectively, exert potent bioactivity, including anticancer effects. This study investigated the antineoplastic activity of these agents against squamous cell carcinoma in vitro and sarcoma 180–induced tumors in vivo. TQ and DG inhibited cell proliferation and induced cytotoxicity in A431 and Hep2 cells. These agents induced apoptosis by increasing the sub-G1 population, LIVE/DEAD cytotoxicity, chromatin condensation, DNA laddering and TUNEL-positive cells significantly (P<0.05). Increased Bax/Bcl-2 ratio, activation of caspases and cleavage of poly ADP ribose polymerase were observed in treated cells. These drugs inhibited Akt and JNK phosphorylations, thus inhibiting cell proliferation while inducing apoptosis. In combination, TQ and DG had synergistic effects, resulting in cell viability as low as 10%. In a mouse xenograft model, a combination of TQ and DG significantly (P<0.05) reduced tumor volume, mass and increased apoptosis. TQ and DG, alone and in combination, inhibit cell proliferation and induce apoptosis in squamous cell carcinoma. The combination of TQ and DG is a potential antineoplastic therapy in this common skin cancer.

Molecular targeting of Akt by thymoquinone promotes G1 arrest through translation inhibition of cyclin D1 and induces apoptosis in breast cancer cells

AIM Thymoquinone (TQ), the predominant bioactive constituent of black seed oil (Nigella Sativa), has been shown to possess antineoplastic activity against multifarious tumors. However, the meticulous mechanism of TQ on Akt mediated survival pathway is still unrevealed in breast cancer. Here, we investigated TQs mechanism of action against PI3K/Akt signaling and its downstream targets by modulating proteins translational machinery, leading to apoptosis in cancer cells. MAIN METHODS MDA-MB-468 and T-47D cells were treated with TQ and evaluated for its anticancer activity through phase distribution and western blot. Modulatory effects of TQ on Akt were affirmed through kinase and drug potential studies. KEY FINDINGS Studies revealed G1 phase arrest till 24h incubation with TQ while extended exposure showed phase shift to subG1 indicating apoptosis, supported by suppression of cyclin D1, cyclin E and cyclin dependent kinase inhibitor p27 expression. Immunoblot and membrane potential studies revealed mitochondrial impairment behind apoptotic process with upregulation of Bax, cytoplasmic cytochrome c and procaspase-3, PARP cleavage along with Bcl-2, Bcl-xL and survivin downregulation. Moreover, we construed the rationale behind mitochondrial dysfunction by examining the phosphorylation status of PDK1, PTEN, Akt, c-raf, GSK-3β and Bad in TQ treated cells, thus ratifying the involvement of Akt in apoptosis. Further, the consequential effect of Akt inhibition by TQ is proven by translational repression through deregulated phosphorylation of 4E-BP1, eIF4E, S6R and p70S6K. SIGNIFICANCE Our observations for the first time may provide a new insight for the development of novel therapies for Akt overexpressed breast cancer by TQ.

Gold nanorod embedded reduction responsive block copolymer micelle-triggered drug delivery combined with photothermal ablation for targeted cancer therapy.

BACKGROUND Gold nanorods, by virtue of surface plasmon resonance, convert incident light energy (NIR) into heat energy which induces hyperthermia. We designed unique, multifunctional, gold nanorod embedded block copolymer micelle loaded with GW627368X for targeted drug delivery and photothermal therapy. METHODS Glutathione responsive diblock co-polymer was synthesized by RAFT process forming self-assembled micelle on gold nanorods prepared by seed mediated method and GW627368X was loaded on to the reduction responsive gold nanorod embedded micelle. Photothermal therapy was administered using cwNIR laser (808nm; 4W/cm2). Efficacy of nanoformulated GW627368X, photothermal therapy and combination of both were evaluated in vitro and in vivo. RESULTS In response to photothermal treatment, cells undergo regulated, patterned cell death by necroptosis. Combining GW627368X with photothermal treatment using single nanoparticle enhanced therapeutic outcome. In addition, these nanoparticles are effective X-ray CT contrast agents, thus, can help in monitoring treatment. CONCLUSION Reduction responsive nanorod embedded micelle containing folic acid and lipoic acid when treated on cervical cancer cells or tumour bearing mice, aggregate in and around cancer cells. Due to high glutathione concentration, micelles degrade releasing drug which binds surface receptors inducing apoptosis. When incident with 808nm cwNIR lasers, gold nanorods bring about photothermal effect leading to hyperthermic cell death by necroptosis. Combination of the two modalities enhances therapeutic efficacy by inducing both forms of cell death. GENERAL SIGNIFICANCE Our proposed treatment strategy achieves photothermal therapy and targeted drug delivery simultaneously. It can prove useful in overcoming general toxicities associated with chemotherapeutics and intrinsic/acquired resistance to chemo and radiotherapy.

BI-69A11 enhances susceptibility of colon cancer cells to mda -7/IL-24-induced growth inhibition by targeting Akt

Background:Akt and its downstream signalling pathways contribute to the aetiology and progression of colorectal carcinoma (CRC). Targeting the Akt pathway is an attractive strategy but few chemotherapeutic drugs have been used to treat CRC with only limited success. BI-69A11, a small molecule inhibitor of Akt, efficiently inhibits growth in melanoma cells. Melanoma differentiation associated gene-7 (mda-7)/interleukin-24 promotes cancer-selective apoptosis when delivered by a tropism-modified replication incompetent adenovirus (Ad.5/3-mda-7). However, Ad.5/3-mda-7 displays diminished antitumour efficacy in several CRC cell lines, which correlates with the expression of K-RAS.Methods:The individual and combinatorial effect of BI-69A11 and Ad.5/3-mda-7 in vitro was studied by cell viability, cell cycle, apoptosis and invasion assays in HT29 and HCT116 cells containing wild type or mutant K-ras, respectively. In vivo HT29 tumour xenografts were used to test the efficacy of the combination treatment.Results:BI-69A11 inhibited growth and induced apoptosis in CRC. However, combinatorial treatment was more effective compared with single treatment. This combination showed profound antitumour and anti angiogenic effects in vitro and in vivo by downregulating Akt activity.Conclusions:BI-69A11 enhances the antitumour efficacy of Ad.5/3-mda-7 on CRC overexpressing K-RAS by inducing apoptosis and regulating Akt activity thereby warranting further evaluation in treating CRC.

Blockade of autophagy enhances proapoptotic potential of BI-69A11, a novel Akt inhibitor, in colon carcinoma

BI-69A11, novel Akt inhibitor, is currently drawing much attention due to its intriguing effect in inducing apoptosis in melanoma, breast, prostate and colon cancer. However, earlier reports reveal that PI3K/Akt/mTOR inhibitors promote autophagy at the early stage as a survival mechanism that might affect its apoptotic potential. It is necessary to investigate whether BI-69A11 mediated apoptosis is associated with autophagy for enhancing its therapeutic efficacy. Here, we found that BI-69A11 induced autophagy at earlier time point through the inhibition of Akt/mTOR/p70S6kinase pathway. Dose-dependent and time-dependent conversion of LC3-I to LC3-II, increased accumulation of LC3-GFP dots in cytoplasm and increase in other autophagic markers such as Beclin-1, firmly supported the fact that BI-69A11 induces autophagy. Atg5, Atg7 and Beclin-1 siRNA mediated genetic attenuation and pre-treatment with pharmacological inhibitor 3-MA and CQ diminished the autophagy and increased the propensity of cell death towards apoptosis. It was also suggested that BI-69A11 mediated interaction between Akt, HSP-90 and Beclin-1 maintained the fine balance between autophagy and apoptosis. Interaction between Beclin-1 and HSP90 is one of the prime causes of induction of autophagy. Here, we also generated a novel combination therapy by pretreatment with CQ that inhibited the autophagy and accelerated the apoptotic potential of BI-69A11. In summary; our findings suggest that induction of autophagy lead to the resistance of colon cancer towards BI-69A11 mediated apoptosis.

Mechanistic attributes of S100A7 (psoriasin) in resistance of anoikis resulting tumor progression in squamous cell carcinoma of the oral cavity

BackgroundSquamous cell carcinoma of the oral cavity (SCCOC) is the dominant origin of cancer associated mortality. Previous findings by our study reported that acquisition of anoikis resistance has a significant role in tumor progression of oral cavity. Several genes were over-expressed in anoikis-resistant cells under detached conditions which we confirmed earlier by microarray. Normal oral squamous epithelia grow adherent to a basement membrane, and when detached from the extracellular matrix, undergoes programmed cell death. The acquisition of anoikis-resistance is crucial phenomena in oral tumor advancement. In the current study, we have identified S100A7 expression as contributing factor for anoikis resistance and tumorigenicity in human oral cancer cells. Further, we have explored that elevated S100A7 expression in anoikis-sensitive oral keratinocytes and cancer cells reshape them more resistant to anoikis and apoptosis inducers via activation of cellular intrinsic and extrinsic avenue.MethodsA subset of human cancer cell lines TU167, JMAR, JMARC39, JMARC42 and MDA-MB-468 were utilized for the generation of resistant stable cell lines. Further, immunohistochemistry, western blot and immunoprecipitation, assays of apoptosis, soft agar assay, orthotopic animal model and signaling elucidation were performed to establish our hypothesis.ResultsS100A7 gene is found to be responsible for anoikis resistance and tumorigenicity in human oral cancer cells. We have observed up-regulation of S100A7 in anoikis resistant cell lines, orthotropic model and patients samples with head and neck cancer. It is also noticed that secretion of S100A7 protein in conditioned medium by anoikis resistant head & neck cancer cell and in saliva of head and neck cancer patients. Up-regulation of S100A7 expression has triggered enhanced tumorigenicity and anchorage-independent growth of cancer cells through Akt phosphorylation leading to development of aniokis resistance in head and neck cancer cells.ConclusionsThese data have led us to conclude that S100A7 is the major contributing factor in mediating anoikis-resistance of oral cancer cells and local tumor progression, and S100A7 might be useful as diagnostic marker for early detection of primary and recurrent squamous cell cancer.

Insights into molecular therapy of glioma: current challenges and next generation blueprint

Glioma accounts for the majority of human brain tumors. With prevailing treatment regimens, the patients have poor survival rates. In spite of current development in mainstream glioma therapy, a cure for glioma appears to be out of reach. The infiltrative nature of glioma and acquired resistance substancially restrict the therapeutic options. Better elucidation of the complicated pathobiology of glioma and proteogenomic characterization might eventually open novel avenues for the design of more sophisticated and effective combination regimens. This could be accomplished by individually tailoring progressive neuroimaging techniques, terminating DNA synthesis with prodrug-activating genes, silencing gliomagenesis genes (gene therapy), targeting miRNA oncogenic activity (miRNA-mRNA interaction), combining Hedgehog-Gli/Akt inhibitors with stem cell therapy, employing tumor lysates as antigen sources for efficient depletion of tumor-specific cancer stem cells by cytotoxic T lymphocytes (dendritic cell vaccination), adoptive transfer of chimeric antigen receptor-modified T cells, and combining immune checkpoint inhibitors with conventional therapeutic modalities. Thus, the present review captures the latest trends associated with the molecular mechanisms involved in glial tumorigenesis as well as the limitations of surgery, radiation and chemotherapy. In this article we also critically discuss the next generation molecular therapeutic strategies and their mechanisms for the successful treatment of glioma.

S100A7 has an oncogenic role in oral squamous cell carcinoma by activating p38/MAPK and RAB2A signaling pathway

Oral cancer consists of squamous cell carcinoma within the oral cavity or on the lip. The clinical prognosis of this cancer is mostly poor owing to delayed diagnosis and a lack of appropriate early detection biomarkers to identify the disease. In the current study, we investigated the role of the S100A7 calcium-binding protein in oral squamous cell carcinoma as an activator of the p38/MAPK and RAB2A signaling pathway. The aim of the present study was to determine whether S100A7 and RAB2A have a role in tumor progression and to assess their potential as early detection biomarkers for oral cancer. This study elucidated the functional and molecular mechanisms of S100A7 and RAB2A activity in oral cancer, leading us to conclude that S100A7 is the major contributing factor in the occurrence of oral cancer and promotes local tumor progression by activating the MAPK signaling pathway via the RAB2A pathway. We hypothesize that S100A7 affects cell motility and invasion by regulating the RAB2A-associated MAPK signaling cascades. Also, the downregulation of S100A7 expression by RNA interference-mediated silencing inhibits oral cancer cell growth, migration and invasion.

Cooperative effect of BI-69A11 and celecoxib enhances radiosensitization by modulating DNA damage repair in colon carcinoma

Amplification of PI3K-Akt pathway promotes radioresistance in various cancers including colorectal carcinoma. Local recurrence in colon cancer causes poor prognosis affecting overall survival of cancer-affected patient population. To avoid local recurrence, pre-operative or post-operative additional radiotherapy is given. However, main concern regarding radiotherapy is to increase the radiosensitivity of malignant cell without hampering the activities of normal cells. In this context, addition of two or more than two chemotherapeutic drugs as a radiosensitizer is a common practice in radiation biology. BI-69A11 earlier showed potential apoptosis-inducing effect in melanoma and colon carcinoma. Celecoxib showed anti-cancer effects in both COX-2 dependent and independent pathways and used to act as a radiosensitizing enhancer. Here, we suggest that the combination of BI-69A11 and celecoxib inhibits the phosphorylation of ataxia telangiectasia mutated (ATM) kinase and DNA-PK responsible for ionizing radiation (IR)-induced double-strand break (DSB) repair. Moreover, the combinatorial effect of BI-69A11 and celecoxib attenuates the IR-induced G2/M cell cycle arrest. Furthermore, this combination also impairs IR-induced activation of Akt and downstream targets of ATM. This might lead to induced activation of apoptotic pathway after triple therapy treatment modulating pro-apoptotic and anti-apoptotic proteins. This activation of apoptotic pathway also showed the interdependence of PUMA and BAD in triple combination-treated colon cancer cells in a p53 independent manner. This study reveals the therapeutic potential of the triple combination therapy in prevention of radioresistance. Besides, it also demonstrates the cytotoxic effects of triple combination therapy in colon cancer. This study shows utility and potential implication on safety of the patients undergoing radiation therapy.