The cytokines in tumor microenvironment: from cancer initiation-elongation-progression to metastatic outgrowth.

It is a well-known fact that cancer can be augmented by infections and inflammation. In fact, chronic inflammation establishes a tumor-supporting-microenvironment (TME), which contributes to neoplastic progression. Presently, extensive research is going on to establish the interrelationship between infection, inflammation, immune response, and cancer. Cytokines are the most essential components in this linkage, which are secreted by immune cells and stromal cells of TME. Cytokines have potential involvement in tumor initiation, elongation, progression, metastatic outgrowth, angiogenesis, and development of therapeutic resistance. They are also linked with increased cancer symptoms along with reduced quality of life in advanced cancer patients. The cancer patients experience multiple symptoms including pain, asthenia, fatigue, anorexia, cachexia, and neurodegenerative disorders etc. Anti-cancer therapeutics can be developed by targeting cytokines along with TME to reduce the immunocompromised state and also modulate the TME. This review article depicts the composition and function of different inflammatory cells within the TME, more precisely the role of cytokines in cancer initiation, elongation, and progression as well as the clinical effects in advanced cancer patients. It also provides an overview of different natural compounds, nanoparticles, and chemotherapeutic agents that can target cytokines along with TME, which finally pave the way for cytokines-targeted anti-cancer therapeutics.

Combination of Resveratrol and PARP inhibitor Olaparib efficiently deregulates homologous recombination repair pathway in breast cancer cells through inhibition of TIP60-mediated chromatin relaxation.

Recently, we reported that a combination of a natural, bioactive compound Resveratrol (RES) and a PARP inhibitor Olaparib (OLA) deregulated the homologous recombination (HR) pathway, and enhanced apoptosis in BRCA1-wild-type, HR-proficient breast cancer cells. Upon DNA damage, chromatin relaxation takes place, which allows the DNA repair proteins to access the DNA lesion. But whether chromatin remodeling has any role in RES + OLA-mediated HR inhibition is not known. By using in vitro and ex vivo model systems of breast cancer, we have investigated whether RES + OLA inhibits chromatin relaxation and thereby blocks the HR pathway. It was found that RES + OLA inhibited PARP1 activity, terminated PARP1-BRCA1 interaction, and deregulated the HR pathway only in the chromatin fraction of MCF-7 cells. DR-GFP reporter plasmid-based HR assay demonstrated marked reduction in HR efficiency in I-SceI endonuclease-transfected cells treated with OLA. RES + OLA efficiently trapped PARP1 at the DNA damage site in the chromatin of MCF-7 cells. Unaltered expressions of HR proteins were found in the chromatin of PARP1-silenced MCF-7 cells, which confirmed that RES + OLA-mediated DNA damage response was PARP1-dependent. Histone Acetyltransferase (HAT) activity and histone H4 acetylation assays showed reduction in HAT activity and H4 acetylation in RES + OLA-treated chromatin fraction of cells. Western blot analysis showed that the HAT enzyme TIP60, P400 and acetylated H4 were downregulated after RES + OLA exposure. In the co-immunoprecipitation assay, it was observed that RES + OLA caused abolition of PARP1-TIP60-BRCA1 interaction, which suggested the PARP1-dependent TIP60-BRCA1 association. Unaltered expressions of PAR, BRCA1, P400, and acetylated H4 in the chromatin of TIP60-silenced MCF-7 cells further confirmed the role of TIP60 in PARP1-mediated HR activation in the chromatin. Similar results were obtained in ex vivo patient-derived primary breast cancer cells. Thus, the present study revealed that RES + OLA treatment inhibited PARP1 activity in the chromatin, and blocked TIP60-mediated chromatin relaxation, which, in turn, affected PARP1-dependent TIP60-BRCA1 association, resulting in deregulation of HR pathway in breast cancer cells.

Nano formulated Resveratrol inhibits PD-L1 in oral cancer cells by deregulating the association between tumor associated macrophages and cancer associated fibroblasts through IL-6/JAK2/STAT3 signaling axis.

Tumor associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs) in the tumor microenvironment secrete several cytokines, which involved in tumor initiation, progression, metastatic outgrowth and angiogenesis. However, the association between TAMs and CAFs in the context of tumor development remain unclear. Here, we studied the relationship between TAMs and CAFs along with the involvement of cytokines in the production of cancer-stem-like-cells (CSCs) in oral cancer cells and explored the potential anticancer effects of Nano-formulated Resveratrol (Res-NP) using an activated macrophage-M1 (AM-M1) and activated fibroblast cells as the model system. IL-6 secretion was found to be enhanced in the conditioned-medium (CM) when AM-M1 cells + CAFs-like cells were cocultured together. CSCs-enriched population was developed after addition of CM of AM-M1 +CAFs in H-357 cells and patient-derived-primary-oral-cancer cells. AM-M1 cells+ CAFs-like cells secreted IL-6 enhanced CSCs growth, proliferation, metastasis, and angiogenesis. IL-6 was found to promote PD-L1 expression in CSCs-enriched cells via JAK2/STAT3 pathway, as evident from the enhanced expression of p-JAK2 and p-STAT3. Nevertheless, Res-NP inhibited CSCs proliferation and reduced the expression of metastatic and angiogenic markers, in ovo blood vascularization, NO production and MMPs expression. Res-NP delinked the association between AM-M1 and CAFs by blocking IL-6 production and also disrupted the potential connection between IL-6 and PD-L1 with considerable decrease in p-JAK2 and p-STAT3 expressions. IL-6 depletion inhibited stemness and angiogenesis in oral CSCs by downregulating PD-L1 via JAK2/STAT3 cascade. Similar observations were also observed in Res-NP treated xenograft mice. Thus, data demonstrate that CSCs growth is dependent on IL-6/PD-L1 axis. Res-NP deregulates the association between AM-M1 and CAFs along-with attenuates carcinogenesis in in vitro, in ovo, ex vivo and in vivo model systems by inhibiting PD-L1 via IL-6/JAK2/STAT3 axis.

Veliparib (ABT-888), a PARP inhibitor potentiates the cytotoxic activity of 5-fluorouracil by inhibiting MMR pathway through deregulation of MSH6 in colorectal cancer stem cells.

OBJECTIVE: Sensitization of mismatch repair (MMR)-deficient colorectal cancer (CRC) cells by 5-Fluorouracil (5-FU) is well-documented. But not much is known about the treatment of MMR-proficient CRC cancer stem cells (CRC-CSCs). Here, we investigated whether a PARP inhibitor (ABT-888) can enhance the 5-FU-mediated apoptosis in CRC-CSCs through MMR pathway inhibition. METHODS: The anti-cancer action of 5-FU+ABT-888 combination in CRC-CSCs has been studied by using in vitro, ex vivo, and in vivo preclinical model systems. RESULTS: 5-FU caused DNA damage in CRC-CSCs, and ABT-888 enhanced the accumulation of DNA mismatches by downregulating the MMR pathway, triggering S-phase arrest, and finally apoptosis and cell death in 5-FU-pre-treated MMR-proficient-CRC-CSCs at much lower concentrations than their individual treatments. After 5-FU treatment, PARylated-PARP1 activated MMR pathway by interacting with MSH6. But, upon ABT-888 treatment in 5-FU-pre-exposed CSCs, PARylation was inhibited, as a result of which PARP1 could not interact with MSH6, and other MMR proteins were downregulated. The role of MSH6 in PARP1-mediated MMR activation, was confirmed by silencing MSH6 gene, which resulted in MMR pathway shutdown. Similar results were obtained in ex vivo and in vivo model systems. CONCLUSIONS: 5-FU+ABT-888 combination enhanced CRC-CSCs death by increasing DNA damage accumulation and simultaneously inhibiting the MMR pathway in MMR-proficient cells. But this study does not discuss whether the combination treatment will increase the sensitivity of MMR-deficient CSCs, for which further research will be performed in the future.

5-FU is a well-known drug commonly used to treat colorectal cancer and it causes DNA damage inside the cancer cells. The limitation of 5-FU treatment is the development of chemoresistance due to the high DNA repair capacity of cancer stem cells present in the tumor microenvironment. In this study, a novel chemotherapeutic approach has been developed to target colorectal cancer stem cells by using a combination of 5-FU and a PARP1 inhibitor (ABT-888). Here, 5-FU caused DNA damage and ABT-888 enhanced the accumulation of the DNA lesions by inhibiting the MMR repair pathway in 5-FU-pre-treated MMR-proficient-CRC-CSCs. This resulted in S-phase arrest, induction of apoptosis, and finally CSCs death.

Quinacrine inhibits HIF-1α/VEGF-A mediated angiogenesis by disrupting the interaction between cMET and ABCG2 in patient-derived breast cancer stem cells.

BACKGROUND: Breast cancer stem cells (BCSCs) have a critical role in progression of breast cancer by inducing angiogenesis. Several therapeutic strategies have been designed for the treatment of breast cancer by specifically preventing angiogenesis. But there is a dearth of study regarding the treatment procedure which can specifically target and kill the BCSCs and cause lesser harm to healthy cells of the body. A plant-based bioactive compound Quinacrine (QC) specifically kills cancer stem cells (CSCs) without harming healthy cells and also inhibits cancer angiogenesis but the detailed mechanistic study of its anti-CSCs and anti-angiogenic activity is yet to explore. HYPOTHESIS: Earlier report showed that both cMET and ABCG2 play an essential role in cancer angiogenesis. Both are present on the cell surface of CSCs and share an identical ATP-binding domain. Interestingly, QC a plant based and bioactive compound which was found to inhibit the function of CSCs marker cMET and ABCG2. These relevant evidence led us to hypothesize that cMET and ABCG2 may interact with each other and induce the production of angiogenic factors, resulting in activation of cancer angiogenesis and QC might disrupt the interaction between them to stop this phenomena. METHODS: Co-immunoprecipitation assay, immunofluorescence assay, and western blotting were performed by using ex vivo patient-derived breast cancer-stem-cells (PDBCSCs) and human umbilical vein endothelial cells (HUVECs). In silico study was carried out to check the interaction between cMET and ABCG2 in presence or absence of QC. Tube formation assay using HUVECs and in ovo Chorioallantoic membrane (CAM) assay using chick fertilized eggs were performed to monitor angiogenesis. In vivo patient-derived xenograft (PDX) mice model was used to validate in silico and ex vivo results. RESULTS: Data revealed that in a hypoxic tumor microenvironment (TME), cMET and ABCG2 interact with each other and upregulate HIF-1α/VEGF-A axis to induce breast cancer angiogenesis. In silico and ex vivo study showed that QC disrupted the interaction between cMET and ABCG2 to inhibit the angiogenic response in endothelial cells by reducing the secretion of VEGF-A from PDBCSCs within the TME. Knockdown of cMET, ABCG2 or both, significantly downregulated the expression of HIF-1α and reduced the secretion of pro-angiogenic factor VEGF-A in the TME of PDBCSCs. Additionally, when PDBCSCs were treated with QC, similar experimental results were obtained. CONCLUSION: In silico, in ovo, ex vivo and in vivo data confirmed that QC inhibited the HIF-1α/VEGF-A mediated angiogenesis in breast cancer by disrupting the interaction between cMET and ABCG2.

Near-infrared enhances antiangiogenic potentiality of quinacrine-gold hybrid nanoparticles in breast cancer stem cells via deregulation of HSP-70/TGF-ß.

Aim: This study aimed to explore the antiangiogenic mechanism of quinacrine-gold hybrid nanoparticle (QAuNP) and near-infrared (NIR) radiation in patient-derived primary breast cancer stem cells. Materials & methods: Various cell-based in ovo angiogenesis and in vivo patient-derived xenograft mouse systems were used as models for the study. Results: The experimental results showed that QAuNP + NIR treatment deregulated the HSP-70/TGF-ß physical interaction in primary breast cancer stem cells. Reduced TGF-ß secretion in the tumor microenvironment inhibited angiogenesis activation in endothelial cells by deregulating the TGF-ß-mediated PI3K/AKT/mTOR cascade. Conclusion: This study revealed that QAuNP + NIR irradiation downregulated HSP-70 expression, inhibited the HSP-70/TGF-ß interaction, reduced the secretion of TGF-ß in the tumor microenvironment and ultimately inhibited TGF-ß-mediated angiogenesis.

This study discovered that the formation of blood vessels in breast cancer is significantly reduced when hybrid nanoparticles and infrared laser therapy are used to treat breast cancer stem cells. The secretory cytokines in the tumor microenvironment primarily responsible for developing blood vessels in the tumor are dramatically reduced by treatment. As a result, the tumor's blood vessel growth is reduced, making it difficult for the cancer cells to get the nutrients and oxygen they need to survive.

Resveratrol nanoparticle attenuates metastasis and angiogenesis by deregulating inflammatory cytokines through inhibition of CAFs in oral cancer by CXCL-12/IL-6-dependent pathway.

Cancer-associated fibroblasts (CAFs) are one of the highly abundant components in the tumor microenvironment (TME). They secrete several cytokines, which amplified tumor progression, invasion, stemness, metastasis, and angiogenesis. Here, we evaluate the potentiality of cytokines for the formation of cancer stem cells (CSCs) in oral cancer cells niche and investigate the anti-inflammatory and anti-carcinogenic effect of Resveratrol-nanoparticle (Res-NP). We first differentiated quiescent human fibroblasts into CAFs in vitro in response to PDGF-B and TGF-ß stimulation and these CAFs were found to increase CXCL-12 and IL-6 secretion. CSCs-enriched population was created by incubating H-357 cells with CAFs and cytokine-enriched CAFs-conditioned media (CAFs-CM). Likewise, CSCs-populated environment was also generated after incubating CAFs-CM to patient-derived primary oral cancer cells. It was noted that CXCL-12 and IL-6 secreted from CAFs significantly promoted CSCs growth, proliferation, aggressiveness, metastasis, and angiogenesis. However, Res-NP reduced CSCs growth and proliferation by abrogating the secretion of CXCL-12 and IL-6. A significant decrease in the expression of metastatic and angiogenic markers, in ovo blood vascularization, intracellular NO generation, MMPs expression and tube formation was found upon Res-NP treatment. Reduction of representative CSCs and angiogenesis markers were also noted after Res-NP treatment in xenograft mice model. CXCL-12 physically interact with IL-6 and this interaction was diminished after Res-NP treatment. Moreover, the expression of CD133 and VEGF-A were down-regulated either on Res-NP or CXCL-12/IL-6-specific inhibitors treated CSCs-enriched cells. Thus, the data suggest that CSCs growth is CXCL-12 and IL-6 dependent and Res-NP obstruct carcinogenesis and metastasis by inhibiting CXCL-12 and IL-6 production in in vitro, in vivo, in ovo, and ex vivo systems.

NIR irradiation enhances the apoptotic potentiality of quinacrine-gold hybrid nanoparticles by modulation of HSP-70 in oral cancer stem cells.

Cancer stem cells (CSCs) are the tumor cell subpopulations that can self-renew, differentiate, initiate and maintain tumor growth. CSCs are frequently drug-resistant, resulting in tumor recurrence, metastasis, and angiogenesis. Herein, using in vitro oral squamous cell carcinoma (OSCC) CSCs and in vivo xenograft mice model, we have systematically studied the apoptotic potentiality of quinacrine-gold hybrid nanoparticle (QAuNP) and its underlying mechanism after NIR irradiation. QAuNP + NIR caused DNA damage and induced apoptosis in SCC-9-CSCs by deregulating mitochondrial membrane potential (ΔΨm) and activation of ROS. Upregulation of CASPASE-3 and DR-5/DR-4 and reduction of heat shock protein (HSP-70) up to 5-fold were also noticed upon the treatment. The increased expression of DR-5 and CASPASE-3 and decreased expression of HSP-70, CD-44 and Ki-67 were also noted in the xenograft mice treated with QAuNP + NIR + TRAIL. Thus, data suggest that the combined treatment enhances apoptosis in OSCC-CSCs by modulating HSP-70 in the DISC.

Nano formulated Resveratrol inhibits metastasis and angiogenesis by reducing inflammatory cytokines in oral cancer cells by targeting tumor associated macrophages.

Tumor associated macrophages in the tumor microenvironment secrete multiple cytokines, which regulate cancer cells growth and invasiveness. We systematically studied the role of cytokines in the induction of cancer stem like cells (CSCs) in oral cancer cells niche and evaluated the mechanism of Resveratrol nanoparticle (Res-Nano) mediated-reduction of CSCs properties in cells. A highly M1-like macrophages-enriched conditioned medium (CM) was generated by treating fixed doses of PMA and LPS in THP-1 cells alone as well as co-cultured of H-357 plus THP-1 cells. These M1-like macrophages increased the production of cytokines (e.g., TNF-α, IL-6, IL-1ß, etc.). A CSCs populated environment was created after addition of cytokine-enriched-CM of co-culture of H-357 and THP-1 cells to cancer cells and cytokine enriched CM of THP-1 cells to patient derived primary oral cancer cells, respectively. After incubation with CM, enhancement of stemness, angiogenic and metastatic properties of both H-357 and primary oral cancer cells were noted. Res-NP decreased the cytokines level in CSCs-enriched cells and reduced the invasion, proliferation and growth of CSCs. Representative metastatic (CD133, ALDH1, CXCR4, etc.) and angiogenic markers (MMPs, iNOS, VEGF-A, etc.) were decreased after Res-NP treatment in CSCs enriched oral cancer cells niche. It also disrupted angiogenesis, depleted nitric oxide production in fertilized chick embryos and reduced the expression of metastatic and angiogenic markers in xenograft mice model system. Thus, this study concluded that CSCs-mediated stemness is a cytokine dependent phenomena and treatment of Res-NP inhibit this process in in vitro, in vivo and ex vivo systems.

Clinical significance of a pvrl 4 encoded gene Nectin-4 in metastasis and angiogenesis for tumor relapse.

PURPOSE: In the present study, we have systematically examined the clinical significance of Nectin-4 (encoded by the PVRL-4 gene), a marker for breast cancer stem cells (CSCs), in cancer metastasis and angiogenesis using a variety of human specimens, including invasive duct carcinoma (IDC) with multiple grades, several types of primary tumors to local and distant relapses, lymph node metastases and circulating tumor cells (CTCs). METHODS: Nectin-4 was overexpressed in more than 92% of samples with 65.2% Nectin-4-positive cells. The level of expression was increased with increasing tumor grade (GI-III) and size (T1-4) of IDC specimens. RESULTS: More induction of Nectin-4 was noted in relapsed samples from a variety of tumors (colon, tongue, liver, kidney, ovary, buccal mucosa) in comparison to primary tumors, while paired adjacent normal tissues do not express any Nectin-4. A high expression of Nectin-4 along with other representative markers in CTCs and lymph node metastasis was also observed in cancer specimens. An increased level of Nectin-4 along with representative metastatic (CD-44, Sca1, ALDH1, Nanog) and angiogenic (Ang-I, Ang-II, VEGF) markers were noted in metastatic tumors (local and distant) in comparison to primary tumors that were correlated with different grades of tumor progression. In addition, greater expression of Nectin-4 was observed in secondary tumors (distant metastasis, e.g., breast to liver or stomach to gall bladder) in comparison to primary tumors. CONCLUSION: Our study demonstrated a significant correlation between Nectin-4 expression and tumor grade as well as stages (p < 0.001), suggesting its association with tumor progression. Nectin-4 was overexpressed at all stages of metastasis and angiogenesis, thus appearing to play a major role in tumor relapse through the PI3K-Akt-NFκß pathway.

PARP inhibitor Olaparib Enhances the Apoptotic Potentiality of Curcumin by Increasing the DNA Damage in Oral Cancer Cells through Inhibition of BER Cascade.

Although Olaparib (Ola, a PARP-inhibitor), in combination with other chemotherapeutic agents, was clinically approved to treat prostate cancer, but cytotoxicity, off-target effects of DNA damaging agents limit its applications in clinic. To improve the anti-cancer activity and to study the detailed mechanism of anti-cancer action, here we have used bioactive compound curcumin (Cur) in combination with Ola. Incubation of Ola in Cur pre-treated cells synergistically increased the death of oral cancer cells at much lower concentrations than individual optimum dose and inhibited the topoisomerase activity. Short exposure of Cur caused DNA damage in cells, but more increased DNA damage was noticed when Ola has incubated in Cur pre-treated cells. This combination did not alter the major components of homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways but significantly altered both short patch (SP) and long patch (LP) base excision repair (BER) components in cancer cells. Significant reduction in relative luciferase activity, expression of BER components and PARylation after Cur and Ola treatment confirmed this combination inhibit the BER activity in cells. Reduction of PARylation, decreased expression of BER components, decreased tumor volume and induction of apoptosis were also noticed in Cur + Ola treated Xenograft mice model. The combination treatment of Cur and Ola also helped in recovering the body weight of tumor-bearing mice. Thus, Cur + Ola combination increased the oral cancer cells death by not only causing the DNA damage but also blocking the induction of BER activity.

Comparative and Mechanistic Study on the Anticancer Activity of Quinacrine-Based Silver and Gold Hybrid Nanoparticles in Head and Neck Cancer.

Using oral cancer cells ( in vitro) and in vivo xenograft mice model, we have systematically studied the detailed mechanism of anticancer activity of quinacrine-based hybrid silver (QAgNP) and gold (QAuNP) nanoparticles (NPs) and compared their efficacies. Both the NPs showed characteristic anti-cell proliferation profile in various cancer cells with minimally affecting the normal nontransformed breast epithelial MCF-10A cells. The IC50 values of QAuNP in various cancer cells were less compared to QAgNP and also found to be the lowest (0.5 µg/mL) in SCC-9 oral cancer cells. Although both NPs caused apoptosis by increased DNA damage, arresting at S phase and simultaneously inhibiting the DNA repair activity in cells, efficacy of QAuNP was better than that of QAgNP. NPs intercalated with DNA and inhibited the topoisomerase activity in cells. Alteration in expression of cell cycle regulatory (cyclins B1, E1, A2, etc.) and replication-related (MRE11, RPA, RFC, etc.) proteins were also observed after NP exposure to the cells. Accumulation of cells resulted in extended G/M phase after prolonged exposure of QAuNP in SCC-9 cells. Interestingly, depletion of geminin and increase of Cdt-1 along with CDC-6 suggest the formation of re-replication. Recovery of body weight and reduction in tumor volume were found in NP-treated xenograft mice. Induction of Bax/Bcl-xL, PARP-1 cleavage, p53, and p21 were noted in NP-treated xenograft mice tissue samples. Thus, data suggest that NP inhibits topoisomerase activity, thereby inhibiting DNA replication and inducing re-replication, which causes S-phase arrest, DNA damage, and finally apoptosis of the oral cancer cells. Also, it was found that anticancer activity of QAuNP is better than that of QAgNP.

Diastereoselective synthesis of novel spiro indanone fused pyrano[3,2-c]chromene derivatives following hetero-Diels-Alder reaction and in vitro anticancer studies.

The development of concise methods for the synthesis of small functionalised spirocyclic molecules is important in the search of new bioactive molecules. To contribute this, here we represent a diastereoselective oxa-hetero-Diels-Alder reaction for the synthesis of novel spiro indanone fused pyrano[3,2-c]chromene derivatives and studied their in vitro anticancer activities. Using previously less explored cyclic ketone i.e. indane-1,3-dione and 3-vinyl-2H-chromene derivatives, we obtained novel spiro-heterocyclic frameworks at the interphase between "drug-like" molecules and natural products. Various spiro indanone fused pyrano[3,2-c]chromene derivatives were synthesized regiospecifically bearing a quaternary stereocenter in high yields (up to 85%) with excellent diastereoselectivity in toluene using 4 Å MS as additive under reflux condition at 120 °C. In vitro cytotoxic studies of these compounds against MCF-7 (breast cancer), HCT-116 (colon cancer), H-357 (oral cancer), MD-MB-231(Breast cancer) cell lines were evaluated by MTT {3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide} assay in vitro. The screening results revealed that many of the compounds are showing moderate to high levels of anticancer activities against the tested cancer cell lines and some displayed potent inhibitory activities in comparison to the commercial anticancer drug 5-fluorouracil (5-FU). Among the series, compound 3'c showed most potent cytotoxicity (15.0-27.5 µM) in three cancer cell lines (MCF-7, HCT-116 and MD-MB-231).