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Cytotoxic chemotherapy alters the immune surface profile of Capan1 and Bxpc3 tumor cells in vitro

Pancreatic cancer is the third most common cause of cancer-related deaths in the United States. Although gemcitabine is the standard of care for most patients with pancreatic cancer, its efficacy is limited by the development of resistance. This resistance may be attributable to the evasion of apoptosis caused by the overexpression of BCL-2 family antiapoptotic proteins. In this study, we investigated the role of BCL-XL in gemcitabine resistance to identify a combination therapy to more effectively treat pancreatic cancer. We used CRISPR-Cas9 screening to identify the key genes involved in gemcitabine resistance in pancreatic cancer. Pancreatic cancer cell dependencies on different BCL-2 family proteins and the efficacy of the combination of gemcitabine and DT2216 (a BCL-XL proteolysis targeting chimera or PROTAC) were determined by MTS, Annexin-V/PI, colony formation, and 3D tumor spheroid assays. The therapeutic efficacy of the combination was investigated in several patient-derived xenograft (PDX) mouse models of pancreatic cancer. We identified BCL-XL as a key mediator of gemcitabine resistance. The combination of gemcitabine and DT2216 synergistically induced cell death in multiple pancreatic cancer cell lines in vitro. In vivo, the combination significantly inhibited tumor growth and prolonged the survival of tumor-bearing mice compared with the individual agents in pancreatic cancer PDX models. Their synergistic antitumor activity is attributable to DT2216-induced degradation of BCL-XL and concomitant suppression of MCL-1 by gemcitabine. Our results suggest that DT2216-mediated BCL-XL degradation augments the antitumor activity of gemcitabine and their combination could be more effective for pancreatic cancer treatment.

Background Patients with advanced T cell lymphomas (TCLs) have limited therapeutic options and poor outcomes in part because their TCLs evade apoptosis through upregulation of anti-apoptotic Bcl-2 proteins. Subsets of TCL cell lines, patient-derived xenografts (PDXs), and primary patient samples depend on Bcl-xL for survival. However, small molecule Bcl-xL inhibitors such as ABT263 have failed during clinical development due to on-target and dose-limiting thrombocytopenia. Methods We have developed DT2216, a proteolysis targeting chimera (PROTAC) targeting Bcl-xL for degradation via Von Hippel-Lindau (VHL) E3 ligase, and shown that it has better anti-tumor activity but is less toxic to platelets compared to ABT263. Here, we examined the therapeutic potential of DT2216 for TCLs via testing its anti-TCL activity in vitro using MTS assay, immunoblotting, and flow cytometry and anti-TCL activity in vivo using TCL cell xenograft and PDX model in mice. Results The results showed that DT2216 selectively killed various Bcl-xL-dependent TCL cells including MyLa cells in vitro. In vivo, DT2216 alone was highly effective against MyLa TCL xenografts in mice without causing significant thrombocytopenia or other toxicity. Furthermore, DT2216 combined with ABT199 (a selective Bcl-2 inhibitor) synergistically reduced disease burden and improved survival in a TCL PDX mouse model dependent on both Bcl-2 and Bcl-xL. Conclusions These findings support the clinical testing of DT2216 in patients with Bcl-xL-dependent TCLs, both as a single agent and in rational combinations.

Small molecules that selectively kill senescent cells (SCs), termed senolytics, have the potential to prevent and treat various age-related diseases and extend healthspan. The use of Bcl-xl inhibitors as senolytics is largely limited by their on-target and dose-limiting platelet toxicity. Here, we report the use of proteolysis-targeting chimera (PROTAC) technology to reduce the platelet toxicity of navitoclax (also known as ABT263), a Bcl-2 and Bcl-xl dual inhibitor, by converting it into PZ15227 (PZ), a Bcl-xl PROTAC, which targets Bcl-xl to the cereblon (CRBN) E3 ligase for degradation. Compared to ABT263, PZ is less toxic to platelets, but equally or slightly more potent against SCs because CRBN is poorly expressed in platelets. PZ effectively clears SCs and rejuvenates tissue stem and progenitor cells in naturally aged mice without causing severe thrombocytopenia. With further improvement, Bcl-xl PROTACs have the potential to become safer and more potent senolytic agents than Bcl-xl inhibitors., Senolytics have the potential to extend healthspan by selectively killing senescent cells (SCs), but senolytics that target Bcl-xl may cause platelet toxicity. Here, the authors generated a Bcl-xl proteolysis-targeting chimera (PROTAC) senolytic, which effectively clears SCs and rejuvenates tissue stem and progenitor cells in naturally aged mice without causing severe thrombocytopenia.

KRAS mutations are the most common oncogenic drivers. Sotorasib (AMG510), a covalent inhibitor of KRAS

B-cell lymphoma extra large (BCL-XL) is a well-validated cancer target. However, the on-target and dose-limiting thrombocytopenia limits the use of BCL-XL inhibitors, such as ABT263, as safe and effective anticancer agents. To reduce the toxicity of ABT263, we converted it into DT2216, a BCL-XL proteolysis-targeting chimera (PROTAC), that targets BCL-XL to the Von Hippel-Lindau (VHL) E3 ligase for degradation. We found that DT2216 was more potent against various BCL-XL-dependent leukemia and cancer cells but considerably less toxic to platelets than ABT263 in vitro because VHL is poorly expressed in platelets. In vivo, DT2216 effectively inhibits the growth of several xenograft tumors as a single agent or in combination with other chemotherapeutic agents, without causing appreciable thrombocytopenia. These findings demonstrate the potential to use PROTAC technology to reduce on-target drug toxicities and rescue the therapeutic potential of previously undruggable targets. Furthermore, DT2216 may be developed as a safe first-in-class anticancer agent targeting BCL-XL. The first BCL-XL-degrading PROTAC achieves safer and more potent antitumor activity than dual BCL-XL and BCL-2 inhibitor navitoclax because of reduced dose-limiting platelet toxicity and high target specificity.

PROteolysis-TArgeting Chimeras (PROTACs) have emerged as an innovative drug development platform. However, most PROTACs have been generated empirically because many determinants of PROTAC specificity and activity remain elusive. Through computational modelling of the entire NEDD8-VHL Cullin RING E3 ubiquitin ligase (CRLVHL)/PROTAC/BCL-xL/UbcH5B(E2)-Ub/RBX1 complex, we find that this complex can only ubiquitinate the lysines in a defined band region on BCL-xL. Using this approach to guide our development of a series of ABT263-derived and VHL-recruiting PROTACs, we generate a potent BCL-xL and BCL-2 (BCL-xL/2) dual degrader with significantly improved antitumor activity against BCL-xL/2-dependent leukemia cells. Our results provide experimental evidence that the accessibility of lysines on a target protein plays an important role in determining the selectivity and potency of a PROTAC in inducing protein degradation, which may serve as a conceptual framework to guide the future development of PROTACs., Simultaneous targeting of BCL-xL and BCL-2 is an attractive approach for cancer treatment. Based on information gained by computational structure modelling, the authors develop a PROTAC that induces degradation of both BCL-xL and BCL-2 and effectively targets BCL-xL/2-dependent leukaemia cells.

BCL-X(L) and BCL-2 are important targets for cancer treatment. BCL-X(L) specific PROTACs have been developed to circumvent the on-target platelet toxicity associated with BCL-X(L) inhibition. However, they have minimal effects on cancer cells that are dependent on BCL-2 or both BCL-X(L) and BCL-2. Here we report a new series of BCL-PROTACs. The lead PZ703b exhibits high potency in inducing BCL-X(L) degradation and in inhibiting but not degrading BCL-2, showing a hybrid dual-targeting mechanism of action that is unprecedented in a PROTAC molecule. As a result, PZ703b is highly potent in killing BCL-X(L) dependent, BCL-2 dependent and BCL-X(L)/BCL-2 dual-dependent cells, in an E3 ligase (VHL)-dependent fashion. We further found that PZ703b forms stable {BCL-2:PROTAC:VCB} ternary complexes in live cells which likely contributes to the enhanced BCL-2 inhibition by PZ703b. With further optimization, analogues of PZ703b could potentially be developed as effective antitumor agents by co-targeting BCL-X(L) and BCL-2.

Core–shell nanostructures are promising platforms for combination drug delivery. However, their complicated synthesis process, poor stability, surface engineering, and low biocompatibility are major hurdles. Herein, a carboxymethyl chitosan-coated poly(lactide-co-glycolide) (cmcPLGA) core–shell nanostructure is prepared via a simple one-step nanoprecipitation self-assembly process. Engineered core–shell nanostructures are tested for combination delivery of loaded docetaxel and doxorubicin in a cancer-mimicked environment. The drugs are compartmentalized in a shell (doxorubicin, Dox) and a core (docetaxel, Dtxl) with loading contents of ∼1.2 and ∼2.06%, respectively. Carboxymethyl chitosan with both amine and carboxyl groups act as a polyampholyte in diminishing ζ-potential of nanoparticles from fairly negative (−13 mV) to near neutral (−2 mV) while moving from a physiological pH (7.4) to an acidic tumor pH (6) that can help the nanoparticles to accumulate and release the drug on-site. The dual-drug formulation was found to carry a clinically comparable 1.7:1 weight ratio of Dtxl/Dox, nanoengineered for the sequential release of Dox followed by Dtxl. Single and engineered combinatorial nanoformulations show better growth inhibition toward three different cancer cells compared to free drug treatment. Importantly, Dox–Dtxl cmcPLGA nanoparticles scored synergism with combination index values between 0.2 and 0.3 in BT549 (breast ductal carcinoma), PC3 (prostate cancer), and A549 (lung adenocarcinoma) cell lines, demonstrating significant cell growth inhibition at lower drug concentrations as compared to single-drug control groups. The observed promising performance of dual-drug formulation is due to the G2/M phase arrest and apoptosis.

BCL-X(L), an anti-apoptotic BCL-2 family protein, plays a key role in cancer cell survival. However, the potential of BCL-X(L) as an anticancer target has been hampered by the on-target platelet toxicity because platelets depend on BCL-X(L) to maintain their viability. Here we report the development of a PROTAC BCL-X(L) degrader, XZ424, which has increased selectivity for BCL-X(L)-dependent MOLT-4 cells over human platelets compared with conventional BCL-X(L) inhibitors. This proof-of-concept study demonstrates the potential of utilizing PROTAC approach to achieve tissue selectivity.

Purpose: Mutations in KRAS are the most common oncogenic drivers. Sotorasib (AMG510), a covalent inhibitor selectively targeting KRASG12C, was recently approved for the treatment of KRASG12C-mutated non-small cell lung cancer (NSCLC). However, the clinical efficacy of sotorasib and other KRASG12C inhibitors is limited by intrinsic resistance in colorectal cancer (CRC) and by the rapid emergence of acquired resistance in all treated solid tumors. Therefore, there is an urgent need to develop novel combination therapies to overcome sotorasib resistance and to maximize its efficacy. Experimental Design: A panel of KRASG12C-mutated cell lines from NSCLC, CRC and pancreatic cancer (PC) were treated with sotorasib alone or in combination with BCL-XL proteolysis targeting chimera (PROTAC, such as DT2216) to assess effects on cell viability, colony formation and apoptosis. To identify mechanisms underlying sotorasib+DT2216 combination responses, we analyzed KRAS downstream signaling and expression of BCL-2 family proteins, and performed co-immunoprecipitation of BCL-XL to evaluate its interaction with pro-apoptotic BH3-only proteins. Finally, therapeutic efficacy of sotorasib alone and in combination with DT2216 was evaluated in vivo using different tumor xenograft models. Results: We observed heterogeneous responses to sotorasib alone, whereas its combination with DT2216 strongly inhibited viability of KRASG12C cell lines that partially responded to sotorasib treatment. Mechanistically, co-treatment with DT2216 inhibited sotorasib-induced BCL-XL/BIM interaction leading to enhanced apoptosis. Furthermore, DT2216 co-treatment significantly improved the antitumor efficacy of sotorasib in vivo. Conclusions: Our findings suggest that due to cytostatic activity, the efficacy of sotorasib is limited, and therefore its combination with a pro-apoptotic agent DT2216 shows synergistic responses and can potentially overcome resistance. Citation Format: Sajid Khan, Janet Wiegand, Peiyi Zhang, Wanyi Hu, Dinesh Thummuri, Vivekananda Budamagunta, Nan Hua, Lingtao Jin, Carmen J. Allegra, Edmund S. Kopetz, Maria Zajac-Kaye, Frederic J. Kaye, Guangrong Zheng, Daohong Zhou. A BCL-XL PROTAC degrader DT2216 synergizes with KRASG12C inhibitors for effectively treating KRASG12C-mutated cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5313.

Pancreatic cancer is the third most common cause of cancer-related deaths in the USA. While gemcitabine (GEM) is the standard-of-care for most pancreatic cancer patients, its efficacy is limited by the development of resistance. This resistance may be attributable to the evasion of apoptosis caused by the overexpression of BCL-2 family anti-apoptotic proteins. In the current study, we investigated the role of BCL-X(L) in GEM resistance to identify a combination therapy to more effectively treat pancreatic cancer. We used CRISPR-Cas9 screening to identify the key genes involved in GEM resistance in pancreatic cancer. Pancreatic cancer cell dependencies on different BCL-2 family proteins and the efficacy of the combination of GEM and DT2216 (a BCL-X(L) proteolysis targeting chimera or PROTAC) were determined by MTS, Annexin-V/PI, colony formation, and 3D tumor spheroid assays. The therapeutic efficacy of the combination was investigated in several patient-derived xenograft (PDX) mouse models of pancreatic cancer. We identified BCL-X(L) as a key mediator of GEM resistance. The combination of GEM and DT2216 synergistically induced cell death in multiple pancreatic cancer cell lines in vitro. In vivo, the combination significantly inhibited tumor growth and prolonged the survival of tumor bearing mice compared with the individual agents in pancreatic cancer PDX models. Their synergistic antitumor activity is attributable to DT2216-induced degradation of BCL-X(L) and concomitant suppression of MCL-1 by GEM. Our results suggest that DT2216-mediated BCL-X(L) degradation augments the antitumor activity of GEM and their combination could be more effective for pancreatic cancer treatment.

Targeting BCL-X(L) via PROTACs is a promising strategy in reducing BCL-X(L) inhibition associated platelet toxicity. Recently, we reported potent BCL-X(L) PROTAC degraders that recruit VHL or CRBN E3 ligase. However, low protein expression or mutation of the responsible E3 ligase has been known to result in decreased protein degradation efficiency of the corresponding PROTACs. To overcome these mechanisms of resistance, PROTACs based on recruiting alternative E3 ligases could be generated. Thus, we designed and synthesized a series of PROTACs that recruit IAP E3 ligases for BCL-X(L) degradation. Among those PROTACs, compound 8a efficiently degrades BCL-X(L) in malignant T-cell lymphoma cell line MyLa 1929 while CRBN-based PROTACs that have high potency in other cancer cell lines show compromised potency, likely due to the low CRBN expression. Moreover, compared with the parent compound ABT-263, PROTAC 8a shows comparable cell killing effects in MyLa 1929 cells whereas the on-target platelet toxicity is significantly reduced. Our findings expand the anti-tumor spectra of BCL-X(L) degraders and further highlight the importance of selecting suitable E3 members to achieve effective cellular activity.

Previous studies in a rat model of Sephadex induced lung inflammation showed that 4-Thiouridine (4SU), a thiol substituted nucleoside, was very effective in reducing edema, leukocyte influx and TNF levels in bronchoalvelolar lavage fluid. However, little is known about the factors and mechanisms underlying these effects. In the present study, we have used two separate mouse models of chronic inflammation, a model of dextran sulphate sodium (DSS) induced colitis and a model of antigen induced arthritis, to evaluate the anti-inflammatory effect of 4-thiouridine. We have analyzed a broad spectrum of inflammatory mediators in order to delineate the mechanisms behind a potential anti-inflammatory effect of 4SU. Colitis was induced in C57BL/6 mice by administration of 3.5% DSS in drinking water for 5 days and the potential anti-colitic effect of 4SU was assessed by monitoring the disease activity index (DAI), measurement of colon length and histopathological analysis of colon tissue. We analyzed tissue myeloperoxidase (MPO) activity, serum pro-inflammatory cytokines (IL-1β, IL-6 and TNF), mRNA and protein expression of pro-inflammatory cytokines, COX-2, and NF-κB activity in colitis tissue. Intracolonic administration of 4SU (5 mg/kg & 10 mg/kg.) significantly inhibited MPO activity and reduced the levels of pro-inflammatory cytokines (IL-1β, IL-6 and TNF) as well as COX-2. Further, NF-κB activation was also blocked by attenuating the phosphorylation of IkB kinase (IKK α/β) in DSS-induced colitis tissues. Arthritis was induced by intra-articular injection of mBSA in the knee of NMRI mice pre-immunized with mBSA and 4SU was administered locally by direct injection into the knee joint. The antiarthritic potential of 4SU was calculated by histopathological scores and histochemical analysis of joint tissue. Further, immunohistochemistry was used to study inflammatory cell infiltration and expression of cytokines and adhesion molecules in the synovium. Local administration of 50–100 mg/kg 4SU at the time of arthritis onset clearly prevented development of joint inflammation and efficiently inhibited synovial expression of CD18, local cytokine production and recruitment of leukocytes to the synovium. Taken together, our data clearly demonstrates a potent anti-inflammatory effect of 4SU in two experimental models. In conclusion 4SU could be a new promising candidate for therapeutic modulation of chronic inflammatory diseases like ulcerative colitis and arthritis.

Anti-apoptotic protein BCL-X(L) plays a key role in tumorigenesis and cancer chemotherapy resistance, rendering it an attractive target for cancer treatment. However, BCL-X(L) inhibitors such as ABT-263 cannot be safely used in the clinic because platelets solely depend on BCL-X(L) to maintain their viability. To reduce the on-target platelet toxicity associated with the inhibition of BCL-X(L), we designed and synthesized PROTAC BCL-X(L) degraders that recruit CRBN or VHL E3 ligase because both of these enzymes are poorly expressed in human platelets compared to various cancer cell lines. We confirmed that platelet-toxic BCL-X(L)/2 dual inhibitor ABT-263 can be converted into platelet-sparing CRBN/VHL-based BCL-X(L) specific degraders. A number of BCL-X(L) degraders are more potent in killing cancer cells than their parent compound ABT-263. Specifically, XZ739, a CRBN-dependent BCL-X(L) degrader, is 20-fold more potent than ABT-263 against MOLT-4 T-ALL cells and has >100-fold selectivity for MOLT-4 cells over human platelets. Our findings further demonstrated the utility of PROTAC technology to achieve tissue selectivity through recruiting differentially expressed E3 ligases.

Autophagy is a homeostatic process that recycles damaged organelles and long-lived proteins by delivering them in double-membrane vesicles to lysosomes for degradation. Autophagy has a prominent role in survival, proliferation, and resistance of tumors in metabolic and chemotherapeutic stress conditions. Clinical trials with chloroquine-a known autophagy inhibitor-were unable to achieve complete autophagy inhibition in vivo, warranting the search for more potent autophagy inhibitors. In a process of exploring the mechanism of action of previously identified cytotoxic s-triazine analogs, we discovered that both IITZ-01 and IITZ-02 act as potent autophagy inhibitors. Treatment with these compounds resulted in the vacuolated appearance of cells due to their specific accumulation in lysosomes. In addition, these basic compounds also deacidify lysosomes as evidenced by the decrease in lysotracker red staining and inhibit maturation of lysosomal enzymes leading to lysosomal dysfunction. IITZ-01 and IITZ-02 enhance autophagosome accumulation but inhibit autophagosomal degradation by impairing lysosomal function, finally resulting in the inhibition of autophagy. Interestingly, compound IITZ-01 exhibited more than 10-fold potent autophagy inhibition along with 12- to 20-fold better cytotoxic action than CQ. IITZ-01 and IITZ-02 also abolished mitochondrial membrane potential and triggered apoptosis through the mitochondria-mediated pathway. Furthermore, IITZ-01 and IITZ-02 displayed potent antitumor action in vivo through autophagy inhibition and apoptosis induction in MDA-MB-231 breast cancer xenograft model with IITZ-01 exhibiting superior anticancer efficacy. Overall, these data demonstrate that IITZ-01 is potent autophagy inhibitor with single-agent anticancer activity and awaits further preclinical development as potential anticancer therapeutic.

Two new series of s-triazine derivatives appended with benzimidazole (15a–h) and benzothiazole derivatives (16a–h) are synthesized, and structure–activity relationships on anticancer activity of these 15a–h and 16a–h were probed. In vitro inhibitory activity against the growth of six cancer cell lines, viz., MCF-7, MDAMB-231, PC-3, DU-145, HT-29 and HGC-27 was evaluated for synthesized analogues. Among the two series of compounds, derivatives containing benzimidazole scaffold were found to be relatively potent over benzothiazole analogues. In accordance with our previous observation, within benzimidazole derivatives, tri-substituted s-triazine derivatives were found to be more potent over di-substituted derivatives irrespective of cell lines. Structure–activity relationships provided useful insights into these classes of compounds and paved the way to design novel analogues with more potency.

Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor, which plays an important role in the cellular defense against oxidative stress by induction of anti-oxidant and cytoprotective enzymes. In the current study, we sought to investigate the osteoprotective effect of carnosic acid (CA), a phenolic (catecholic) diterpene. It is widely identified for its electrophilic nature under oxidative stress conditions and thus anticipated to counter osteoporosis by facilitation of Nrf2 signalling. Osteoclast differentiation was induced by incubation of RAW 264.7 (mouse macrophage) cells and mouse bone marrow macrophages (BMMs) in the presence of receptor activator of NF-κB ligand (RANKL) (100 ng/ml). After treatment, osteoclastogenesis was assessed using tartrate-resistant acid phosphatase (TRAP) assay. We observed that 6 h pretreatment with CA (1.25, 2.5, 5 μM) decreased RANKL-induced osteoclast formation and abolished RANKL-induced ROS generation by activating Nrf2 and its transcriptional targets. Further, CA also inhibited RANKL-induced activation of NF-κB and MAPK signalling. RANKL-induced mRNA expression of osteoclast related genes and transcription factors was also diminished by CA. In vivo osteolysis was developed in C57BL/6 male mice using lipopolysaccharide (LPS). Consistent with in vitro results, in vivo μ-CT analysis of femurs showed that bone mineral density (BMD), bone mineral content (BMC), and bone architecture parameters such as trabecular thickness (Tb.Th) and trabecular space (Tb.Sp) were positively modulated by CA in LPS-injected mice. The results obtained in this study support that CA inhibits RANKL-induced osteoclastogenesis by maintaining redox homeostasis through modulation of Nrf2 and NF-κB pathways.Carnosic acid (CA) inhibits RANKL-induced osteoclastogenesis. CA inhibits RANKL-induced oxidative stress by upregulating Nrf2 transcriptional targets. CA attenuates RANKL-induced NF-κB and MAPK signalling activation. CA decreases NFATc1 and c-Fos expression. In vivo μ-CT analysis reveals that CA prevents bone loss in LPS-injected mice.

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related death with a median survival time of 6–12 months. Most patients present with disseminated disease and the majority are offered palliative chemotherapy. With no approved treatment modalities for patients who progress on chemotherapy, we explored the effects of long-term gemcitabine administration on the tumor microenvironment to identify potential therapeutic options for chemorefractory PDAC. Using a combination of mouse models, primary cell line–derived xenografts, and established tumor cell lines, we first evaluated chemotherapy-induced alterations in the tumor secretome and immune surface proteins by high throughput proteomic arrays. In addition to enhancing antigen presentation and immune checkpoint expression, gemcitabine consistently increased the synthesis of CCL/CXCL chemokines and TGFβ-associated signals. These secreted factors altered the composition of the tumor stroma, conferring gemcitabine resistance to cancer-associated fibroblasts in vitro and further enhancing TGFβ1 biosynthesis. Combined gemcitabine and anti-PD-1 treatment in transgenic models of murine PDAC failed to alter disease course unless mice also underwent genetic or pharmacologic ablation of TGFβ signaling. In the setting of TGFβ signaling deficiency, gemcitabine and anti-PD-1 led to a robust CD8+ T-cell response and decrease in tumor burden, markedly enhancing overall survival. These results suggest that gemcitabine successfully primes PDAC tumors for immune checkpoint inhibition by enhancing antigen presentation only following disruption of the immunosuppressive cytokine barrier. Given the current lack of third-line treatment options, this approach warrants consideration in the clinical management of gemcitabine-refractory PDAC.Significance:These data suggest that long-term treatment with gemcitabine leads to extensive reprogramming of the pancreatic tumor microenvironment and that patients who progress on gemcitabine-based regimens may benefit from multidrug immunotherapy.See related commentary by Carpenter et al., p. 3070

Radiation-induced pulmonary fibrosis (RIPF) is a serious treatment complication that affects about 9%-30% cancer patients receiving radiotherapy for thoracic tumors. RIPF is characterized by progressive and irreversible destruction of lung tissues and deterioration of lung function, which can compromise quality of life and eventually lead to respiratory failure and death. Unfortunately, the mechanisms by which radiation causes RIPF have not been well established nor has an effective treatment for RIPF been developed. Recently, an increasing body of evidence suggests that induction of senescence by radiation may play an important role in RIPF and clearance of senescent cells (SnCs) with a senolytic agent, small molecule that can selectively kill SnCs, has the potential to be developed as a novel therapeutic strategy for RIPF. This review discusses some of these new findings to promote further study on the role of cellular senescence in RIPF and the development of senolytic therapeutics for RIPF.

BCL-XL is a well-validated cancer target. However, the on-target and dose-limiting thrombocytopenia limits the use of BCL-XL inhibitors such as ABT263 as safe and effective anticancer agents. To reduce the toxicity of ABT263, we converted it into DT2216, a BCL-XL proteolysis targeting chimera (PROTAC), that targets BCL-XL to the Von Hippel-Lindau (VHL) E3 ligase for degradation. We found that DT2216 was more potent against various BCL-XL-dependent leukemia and cancer cells but significantly less toxic to platelets than ABT263 in vitro because VHL is poorly expressed in platelets. In vivo, DT2216 effectively inhibits the growth of several xenograft tumors as a single agent or in combination with other chemotherapeutic agents, without causing significant thrombocytopenia. These findings demonstrate the potential to use PROTAC technology to reduce on-target drug toxicities and rescue the therapeutic potential of previously undruggable targets. Furthermore, DT2216 may be developed as a safe first-in-class anticancer agent targeting BCL-XL.

A new series of (E)-benzo[d]imidazol-2-yl)methylene)indolin-2-one derivatives has been synthesized and evaluated for their in vitro cytotoxic activity against a panel of selected human cancer cell lines of prostate (PC-3 and DU-145) and breast (BT-549, MDA-MB-231, MCF-7, 4T1), non-small lung (A549) and gastric (HGC) cancer cells along with normal breast epithelial cells (MCF10A). Among the tested compounds, 8l showed significant cytotoxic activity against MDA-MB-231 and 4T1 cancer cells with IC50 values of 3.26 ± 0.24 μM and 5.96 ± 0.67 μM respectively. The compounds 8f, 8i, 8l and 8o were also screened on normal human breast epithelial cells (MCF10A) and found to be safer with lesser cytotoxicity. The treatment of MDA-MB-231 cells with 8l led to inhibition of cell migration ability through disruption of F-actin protein assembly. The flow-cytometry analysis reveals that the cells arrested in G0/G1 phase of the cell cycle. Further, the compound 8l induced apoptosis of MDA-MB-231 cells was characterized by different staining techniques such as Acridine Orange/Ethidium Bromide (AO/EB), DAPI, annexin V-FITC/PI, Rhodamine-123 and MitoSOX red assay. Western blot studies demonstrated that the compound 8l treatment led to activation of caspase-3, increased expression of cleaved PARP, increased expression of pro-apoptotic Bax and decreased expression of anti-apoptotic Bcl-2 in MDA-MB-231 cancer cells.

Cardamonin (CD), an active chalconoid, has shown potent anticancer effects in preclinical studies; however, the effect and underlying mechanism of CD for the treatment of triple negative breast cancer (TNBC) is unclear. This study aims to examine the cytotoxic effects of CD and investigate the underlying mechanism in human TNBC cells. The results show that CD exhibits cytotoxicity by inducing apoptosis and cell cycle arrest in TNBC cells via modulation of Bcl-2, Bax, cyt-C, cleaved caspase-3, and PARP. We find that CD significantly increases expression of the epithelial marker E-cadherin, while reciprocally decreasing expression of mesenchymal markers such as snail, slug, and vimentin in BT-549 cells. In parallel with epithelial-mesenchymal transition (EMT) reversal, CD down regulates invasion and migration of BT-549 cells. CD markedly reduces stability and nuclear translocation of β-catenin, accompanied with downregulation of β-catenin target genes. Using the TopFlash luciferase reporter assay, we reveal CD as a specific inhibitor of the Wnt3a-induced signaling. These results suggest the involvement of the Wnt/β-catenin signaling in the CD-induced EMT reversion of BT-549 cells. Notably, CD restores the glycogen synthase kinase-3β (GSK3β) activity, required for β-catenin destruction via the proteasome-mediated system, by inhibiting the phosphorylation of GSK3β by Akt. These occurrences ultimately lead to the blockage of EMT and the invasion of TNBC cells. Further antitumor activity of CD was tested in 4T1 (TNBC cells) induced tumor and it was found that CD significantly inhibited the tumor volume at dose of 5 mg/kg-treated mice. © 2016 BioFactors, 43(2):152-169, 2017.

A series of new (Z)-3-(3'-methoxy-4'-(2-amino-2-oxoethoxy)benzylidene)indolin-2-one derivatives has been synthesized and evaluated for their cytotoxic activity against selected human cancer cell lines of prostate (PC-3 and DU-145), breast (BT-549 and MDA-MB-231) and non-tumorigenic prostate epithelial cells (RWPE-1). Among the tested, one of the compounds 4p exhibited potent cytotoxicity selectively on prostate cancer cell lines (PC-3 and DU-145; IC50: 1.89 ± 0.6 and 1.94 ± 0.2 μM, respectively). Further experiments were conducted with 4p on PC-3 cancer cells to study the mechanisms of growth inhibition and apoptosis inducing effect. Treatment of PC-3 cells with test compound 4p resulted in inhibition of cell migration through disorganization of F-actin protein. The flow-cytometry analysis results showed that the compound arrested PC-3 cancer cells in the G2/M phase of cell cycle in a dose dependent manner. Hoechst staining and annexin-V binding assay revealed that the compound 4p inhibited tumor cell proliferation through induction of apoptosis. Western blot studies demonstrated that the compound 4p treatment led to activation of caspase-3, increased expression of pro-apoptotic Bax and significantly decreased expression of anti-apoptotic Bcl-2 in human prostate cancer PC-3 cells. In addition, the mitochondrial membrane potential (ΔΨm) was also affected and the levels of intracellular Ca(2+) were raised.

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related death with a median survival time of 6-12 months. As most patients present with disseminated disease, the majority are offered palliative chemotherapy. With no approved treatment modalities for patients who progress on chemotherapy, we explored the effects of long-term Gemcitabine on the tumor microenvironment in order to identify potential therapeutic options in chemo-refractory PDAC. Using a combination of mouse models, patient-derived xenografts, primary and established cell lines, we first evaluated chemotherapy-induced alterations in the tumor secretome and immune surface proteins by high throughput proteomic arrays. In addition to enhancing antigen presentation and immune checkpoint expression, Gemcitabine consistently increased the synthesis of CCL/CXCL chemokines and TGFβ-associated signals. These secreted factors altered the composition of the tumor stroma, conferring Gemcitabine-resistance to cancer-associated fibroblasts in vitroand further enhancingTGFβ1 biosynthesis. We therefore combined Gemcitabine with anti-PD-1 in transgenic models of murine PDAC, which failed to alter disease course unless mice also underwent genetic or pharmacologic ablation of TGFβ signaling. In the setting of TGFβ signal deficiency, Gemcitabine and anti-PD-1 led to a robust CD8+ T-cell response and decrease in tumor burden, markedly enhancing overall survival. These results suggest that Gemcitabine may prime PDAC tumors for immune checkpoint inhibition by enhancing antigen presentation, but requires the inhibition of an immunosuppressive cytokine barrier. Given the current lack of third line treatment options, this approach warrants consideration in the clinical management of Gemcitabine-refractory PDAC. Citation Format: Daniel R. Principe, Matthew Narbutis, Sandeep Kumar, Alex Park, Navin Viswakarma, Matthew J. Dorman, Suneel D. Kamath, Paul Grippo, Melissa L. Fishel, Rosa F. Hwang, Dinesh Thummuri, Patrick W. Underwood, Hidayatullah G. Munshi, Jose G. Trevino, Ajay Rana. Long-term gemcitabine treatment reshapes the pancreatic tumor microenvironment and sensitizes murine carcinoma to combination immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 955.

Targeting BCL-XL via PROTACs is a promising strategy in reducing BCL-XL inhibition associated platelet toxicity. Recently, we reported potent BCL-XL PROTAC degraders that recruit VHL or CRBN E3 ligase. However, low protein expression or mutation of the responsible E3 ligase has been known to result in decreased protein degradation efficiency of the corresponding PROTACs. To overcome these mechanisms of resistance, PROTACs based on recruiting alternative E3 ligases could be generated. Thus, we designed and synthesized a series of PROTACs that recruit IAP E3 ligases for BCL-XL degradation. Among those PROTACs, compound 8a efficiently degrades BCL-XL in malignant T-cell lymphoma cell line MyLa 1929 while CRBN-based PROTACs that have high potency in other cancer cell lines show compromised potency, likely due to the low CRBN expression. Moreover, compared with the parent compound ABT-263, PROTAC 8a shows comparable cell killing effects in MyLa 1929 cells whereas the on-target platelet toxicity is significantly reduced. Our findings expand the anti-tumor spectra of BCL-XL degraders and further highlight the importance of selecting suitable E3 members to achieve effective cellular activity.

Autophagy is a homeostatic process that recycles damaged organelles and long-lived proteins by delivering them in double-membrane vesicles to lysosomes for degradation. Autophagy has a prominent role in survival, proliferation, and resistance of tumors in metabolic and chemotherapeutic stress conditions. Clinical trials with chloroquine-a known autophagy inhibitor-were unable to achieve complete autophagy inhibition in vivo, warranting the search for more potent autophagy inhibitors. In a process of exploring the mechanism of action of previously identified cytotoxic s-triazine analogs, we discovered that both IITZ-01 and IITZ-02 act as potent autophagy inhibitors. Treatment with these compounds resulted in the vacuolated appearance of cells due to their specific accumulation in lysosomes. In addition, these basic compounds also deacidify lysosomes as evidenced by the decrease in lysotracker red staining and inhibit maturation of lysosomal enzymes leading to lysosomal dysfunction. IITZ-01 and IITZ-02 enhance autophagosome accumulation but inhibit autophagosomal degradation by impairing lysosomal function, finally resulting in the inhibition of autophagy. Interestingly, compound IITZ-01 exhibited more than 10-fold potent autophagy inhibition along with 12- to 20-fold better cytotoxic action than CQ. IITZ-01 and IITZ-02 also abolished mitochondrial membrane potential and triggered apoptosis through the mitochondria-mediated pathway. Furthermore, IITZ-01 and IITZ-02 displayed potent antitumor action in vivo through autophagy inhibition and apoptosis induction in MDA-MB-231 breast cancer xenograft model with IITZ-01 exhibiting superior anticancer efficacy. Overall, these data demonstrate that IITZ-01 is potent autophagy inhibitor with single-agent anticancer activity and awaits further preclinical development as potential anticancer therapeutic.

A series of new β-carboline-dithiocarbamate derivatives bearing phenyl, dithiocarbamate and H/methyl substitutions at position-1, 3 and 9, respectively, were designed and synthesized. These derivatives 8a-l and 13a-l and their starting precursors (7 a-d and 12 a-d) have been evaluated for their in vitro cytotoxic activity on selected human cancer cell lines. Among the derivatives tested, 7 c, 12 c, 8 a, 8 d, 8 i, 8 j, 8 k, 8l and 13 d-l exhibited considerable cytotoxicity against most of the tested cancer cell lines (IC50

Emu (Dromaius novaehallandiae), the flightless bird native to Australia and found in many countries, is receiving much attention for its nutritional benefits as well as its medicinal value. Emu oil contains high amounts of polyunsaturated fatty acids and antioxidants. It has potent anti-inflammatory actions and thus can be used topically and orally to treat conditions such as mucositis, inflammatory bowel syndrome, and auricular inflammation, and to prevent chemotherapy-induced bone loss. Emu oil also has a hypocholesterolemic effect, transdermal penetration-enhancing activity, cosmetic and insect repellent activity, and so on. However, its mechanism(s) of actions are unclear and have not, to our knowledge, been studied to date. Previous studies suggest that the fatty acids of the ω-9, ω-6, and ω-3 series, which are present in emu oil, may act on cyclooxygenase, lipoxygenase, and lipoxin pathways to bring about its anti-inflammatory and other beneficial actions. The aim of this review was to provide a brief summary of the current knowledge of research on emu products, mainly emu oil, for the possible use as a complementary and alternative natural medicine for various chronic diseases. In this review we also highlighted the future research scope of emu oil for its possible antidiabetic activity. Thus, emu oil is an attractive pharmacologic agent to further explore for its therapeutic activity to treat various ailments.

A total of twenty-two novel coumarin triazole hybrids (4a-4k and 6a-6k) were synthesized from orcinol in good to excellent yields of 70–94%. The structures of all the synthesized compounds were elucidated by spectroscopic techniques such as 1H NMR, 13C NMR, and HRMS. The anti-inflammatory potential of synthesized compounds was investigated against the proinflammatory cytokine, TNF-α on U937 cell line and compounds 4d, 4j, and 6j were found to exhibit promising anti-inflammatory activity. These three compounds were further screened against TNF-α on LPS-stimulated RAW 264.7 cells, which confirm their anti-inflammatory potential. Furthermore, the above said active compounds were tested for their inhibitory effect on RANKL-induced osteoclastogenesis in RAW 264.7 cells by using tartrate resistant acid phosphatase (TRAP) staining assay at 10 µM. Molecular mechanism studies demonstrated that compound 4d exhibited dose dependent inhibition of RANKL-induced osteoclastogenesis by suppression of the NF-kB pathway. Thus, compound 4d is a promising candidate for further optimization to develop as a potent anti-osteoporotic agent.

Osteoporosis is a major debilitating cause of fractures and decreases the quality of life in elderly patients. Bone homeostasis is maintained by bone forming osteoblasts and bone resorpting osteoclasts. Substantial evidences have shown that targeting osteoclasts using natural products is a promising strategy for the treatment of osteoporosis. In the current study, we investigated the osteoprotective effect of Abietic acid (AA) in in vitro and in vivo models of osteolysis. In vitro experiments demonstrated that, AA suppressed receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis and F-actin ring formation in a concentration dependent manner. Mechanistically, AA abrogated RANKL-induced phosphorylation of IKKα/β (ser 176/180), IkBα (ser 32), and inhibited the nuclear translocation of NF-κB. We also found that, AA attenuated the RANKL-induced phosphorylation of MAPKs and decreased the expression of osteoclast specific genes such as TRAP, DC-STAMP, c-Fos, and NFATc1. Consistent with in vitro results, in vivo Lipoploysaccharide (LPS)-induced osteolysis model showed that AA inhibited the LPS-induced serum surge in cytokines TNF-α and IL-6. μ-CT analysis showed that AA prevented the LPS-induced osteolysis. Furthermore, histopathology and TRAP staining results suggested that AA decreased the number of osteoclasts in LPS-injected mice. Taken together, we demonstrated that the osteoprotective action of AA is coupled with the inhibition of NF-κB and MAPK signaling and subsequent inhibition of NFATc1 and c-Fos activities. Hence, AA may be considered as a promising drug candidate for the treatment of osteoporosis.

Several rhodanine derivatives ( 9 – 39 ) were synthesized for evaluation of their potential as anticancer agents. Villsmeier cyclization to synthesize aza-aromatic aldehydes, rhodanine derivatives preparation and Knoevenagel type of condensation between the rhodanines and aza-aromatic aldehydes are key steps used for the synthesis of 31 compounds. In vitro antiproliferative activity of the synthesized rhodanine derivatives ( 9 – 39 ) was studied on a panel of six human tumor cell lines viz. HGC, MNK-74, MCF-7, MDAMB-231, DU-145 and PC-3 cell lines. Some of the compounds were capable of inhibiting the proliferation of cancer cell lines at a micromolar concentration. Six compounds are found to be potent against HGC cell lines; compound 15 is found to be active against HGC – Gastric, MCF7 – Breast Cancer and DU145 – Prostate Cancer cell lines; compound 39 is potent against MNK-74; four compounds are found to be potent against MCF-7 cell lines; three compounds are active against MDAMB-231; nine compounds are found to be potent against DU-145; three compounds are active against PC-3 cell lines. These compounds constitute a promising starting point for the development of novel and more potent anticancer agents in future.

A series of new benzimidazole-thiazolidinedione hybrids has been synthesized and evaluated for their cytotoxic potential against a selected human cancer cell lines of prostate (PC-3 and DU-145), breast (MDA-MB-231), lung (A549) and a normal breast epithelial cells (MCF10A). Among the tested compounds, 11p exhibited promising cytotoxicity with IC50 value of 11.46 ± 1.46 μM on A549 lung cancer cell line and did not show significant toxicity on normal MCF10A cells. Lung cancer cells (A549) have been used to know the mechanism of cell growth inhibition and apoptosis inducing effect with compound 11p. The treatment of A549 cells with 11p showed typical apoptotic morphology like cell shrinkage, chromatin condensation and horseshoe shaped nuclei formation. Flow-cytometry analysis revealed the G2/M phase of cell cycle arrest in a dose dependent manner. Preliminary mechanistic studies suggested that the cell migration was inhibited through the disruption of F-actin protein. Acridine orange-ethidium bromide (AO-EB), DAPI, annexin V-FITC/propidium iodide, rhodamine-123 and MitoSOX assays suggested the induction of apoptosis in A549 cells by compound 11p.

A series of new 4′- O -alkylamino-tethered-benzylideneindolin-2-one derivatives has been synthesized and evaluated for their anti-proliferative activity against selected human cancer cell lines of lung (A549), prostate (DU-145), breast (BT549 and MDA-MB-231) and normal breast epithelial cells (MCF-10A). Gratifyingly, the compounds 5j , 5o and 5r exhibited potent cytotoxicity against breast cancer cell lines (BT549 and MDA-MB-231) with IC 50 values in the range of 1.26–2.77 μM, and are found to be safer with lesser cytotoxicity on normal breast epithelial cells (MCF-10A). Further, experiments were conducted with these compounds 5j , 5o and 5r on MDA-MB-231 cancer cells to study the mechanism of growth inhibition and apoptosis inducing effect. Treatment of MDA-MB-231 cells with test compounds resulted in inhibition of cell migration through disorganization and disruption of F-actin capping protein. The flow-cytometry analysis results showed that the compound 5o arrested MDA-MB-231 cells in G0/G1 phase of cell cycle in a dose dependent manner. Hoechst staining study revealed that the test compounds inhibited tumor cell proliferation through induction of apoptosis. In addition, the mitochondrial membrane potential (DΨm) was affected and the increased level of reactive oxygen species (ROS) was noted in MDA-MB-231 cells.

The evasion of apoptosis, or programmed cell death, is a hallmark of cancer, which promotes tumor initiation and progression. The evasion is in part attributable to the over-expression of anti-apoptotic proteins in the Bcl-2 family. In addition, chemotherapy and radiation can upregulate the expression of the Bcl-2 family in cancer cells, which renders them more resistance to cancer therapy. The most common Bcl-2 family member over-expressed in many solid tumor cells and a fraction of leukemia and lymphoma cells is Bcl-XL and its expression is also highly correlated with resistance to cancer therapy independent of p53 status in many cancers. Therefore, Bcl-XL is one of the most important validated cancer cell targets. Inhibition of Bcl-XL with a small molecule inhibitor has been extensively exploited as a molecularly targeted therapeutic strategy against cancer, resulting in the discovery of several Bcl-2/XL and Bcl-XL inhibitors as promising anti-cancer drug candidates including navitoclax. Unfortunately, these inhibitors failed to become anticancer drugs because platelets are also dependent on Bcl-XL for survival. Therefore, inhibition of Bcl-XL with Bcl-2/XL and Bcl-XL inhibitors causes severe reduction in platelets or thrombocytopenia, an on-target and dose-limiting toxicity, which prevents their use as an effective anticancer drug in clinic. To overcome this problem, we generated a series of novel bifunctional molecules that targeting Bcl-XL to the ubiquitin-proteasome system (UPS) for degradation. These synthetic proteolytic compounds, termed synthetic proteolytics (Syntholytics) or proteolysis targeting chimeras (PROTACs), were rationally designed to recruit the Von Hippel Lindau (VHL) E3 ligase to ubiquitinate Bcl-XL for degradation by the proteasome. Because VHL is minimally expressed in platelets, our Bcl-XL Syntholytics can selectively induce Bcl-XL degradation in various cancer cells but not in platelets. Amongst these Bcl-XL Syntholytics, DT2216 was found to be the most potent in inducing Bcl-XL degradation leading to the loss of viability of Bcl-XL-dependent T-ALL MOLT-4 cells at nanomolar concentrations but did not cause any platelet toxicity. Compared to navitoclax, DT2216 is more potent in induction of apoptosis in a variety of cancer and leukemia cells in vitro in a caspase-dependent manner. Furthermore, our in vivo studies in immunocompromised mice revealed that DT2216 at 15 mg/kg/wk potently inhibited tumor growth in Bcl-XL-dependent MOLT-4 T-ALL xenografts as a single agent whereas navitoclax had no significant effect at the same dosage. Dosing with DT2216 at 15 mg/kg every four days significantly regressed larger established MOLT-4 T-ALL tumors that failed to respond to navitoclax treatment. To assess the therapeutic potential of DT2216 in combination with other Bcl-2 family inhibitors, we employed the Bcl-2/xl dependent NCI-H146 small cell lung cancer cells and the Mcl1/Bcl-xl dependent multiple myeloma EJM cells. The combination of DT2216 with Bcl-2 inhibitor (ABT199) or Mcl-1 inhibitor (S63845) synergistically reduced the viability of H146 and EJM cells, respectively. DT2216 in combination with ABT199 effectively inhibited tumor growth in H146 xenografts. Collectively, our findings suggest that targeting Bcl-XL using Bcl-XL Syntholytics can selectively kill Bcl-XL-dependent T-ALL cells and various solid tumor cells without causing significant platelet toxicity. Moreover, the combination of Bcl-XL Syntholytics with other Bcl-2 protein inhibitors could be used to effectively target multiple cancer types including both hematological and solid tumors. Therefore, Bcl-XL Syntholytics have the potential to be developed as safer and more potent novel anti-cancer drugs. Keywords: Bcl-XL, VHL, Protein degradation, T-ALL, Cancer, Apoptosis Disclosures: S.K., X.Z., D.L., Y.H., P.Z., X. L., G. Z., and D.Z. are inventors of a pending patent application for use of Bcl-xl syntholytics as anti-cancer agents. R.H, G.Z. and D.Z. are co-founders of Dialectic Therapeutics that develops Bcl-xl syntholytics. Disclosures Khan: Dialectic Therapeutics: Patents & Royalties. Lv:Dialectic Therapeutics: Patents & Royalties. He:Dialectic Therapeutics: Patents & Royalties. Zhang:Dialectic Therapeutics: Patents & Royalties. Liu:Dialectic Therapeutics: Patents & Royalties. Konopleva:Stemline Therapeutics: Research Funding. Zheng:Dialectic Therapeutics: Consultancy, Equity Ownership, Patents & Royalties.

Osteoclasts are multinuclear giant cells responsible for bone resorption in inflammatory bone diseases such as osteoporosis, rheumatoid arthritis and periodontitis. Because of deleterious side effects with currently available drugs the search continues for novel effective and safe therapies. Thymoquinone (TQ), the major bioactive component of Nigella sativa has been investigated for its anti-inflammatory, antioxidant and anticancer activities. However, its effects in osteoclastogenesis have not been reported. In the present study we show for the first time that TQ inhibits nuclear factor-KB ligand (RANKL) induced osteoclastogenesis in RAW 264.7 and primary bone marrow derived macrophages (BMMs) cells. RANKL induced osteoclastogenesis is associated with increased expression of multiple transcription factors via activation of NF-KB, MAPKs signalling and reactive oxygen species (ROS). Mechanistically TQ blocked the RANKL induced NF-KB activation by attenuating the phosphorylation of IkB kinase (IKKα/β). Interestingly, in RAW 264.7 cells TQ inhibited the RANKL induced phosphorylation of MAPKs and mRNA expression of osteoclastic specific genes such as TRAP, DC-STAMP, NFATc1 and c-Fos. In addition, TQ also decreased the RANKL stimulated ROS generation in macropahges (RAW 264.7) and H2O2 induced ROS generation in osteoblasts (MC-3T3-E1). Consistent with in vitro results, TQ inhibited lipopolysaccharide (LPS) induced bone resorption by suppressing the osteoclastogenesis. Indeed, micro-CT analysis showed that bone mineral density (BMD) and bone architecture parameters were positively modulated by TQ. Taken together our data demonstrate that TQ has antiosteoclastogenic effect by inhibiting inflammation induced activation of MAPKs, NF-KB and ROS generation followed by suppressing the gene expression of c-Fos and NFATc1 in osteoclast precursors.

There has been a search for new anticancer agents to treat cancer resistance throughout the globe. Salinomycin (SAL), a broad spectrum antibiotic and a coccidiostat has been found to counter tumour resistance and kill cancer stem cells with better efficacy than the existing chemotherapeutic agents; paclitaxel and doxorubicin. This refocused its importance for treatment of human cancers. In this study, we studied the in vitro drug metabolism and pharmacokinetic parameters of SAL. SAL undergoes rapid metabolism in liver microsomes and has a high intrinsic clearance. SAL metabolism is mainly mediated by CYP enzymes; CYP3A4 the major enzyme metabolising SAL. The percent plasma protein binding of SAL in human was significantly lower as compared to mouse and rat plasma. CYP inhibition was carried out by chemical inhibition and recombinant enzyme studies. SAL was found to be a moderate inhibitor of CYP2D6 as well as CYP3A4. As CYP3A4 was the major enzyme responsible for metabolism of SAL, in vivo pharmacokinetic study in rats was done to check the effect of concomitant administration of Ketoconazole (KTC) on SAL pharmacokinetics. KTC, being a selective CYP3A4 inhibitor increased the systemic exposure of SAL significantly to 7-fold in AUC0-α and 3-fold increase in Cmax of SAL in rats with concomitant KTC administration.

The present study was aimed to evaluvate the apoptogenic potential of ethanolic extract of leaves from Boswellia ovalifoliolata (BL EthOH) and to unravel the molecular mechanisms implicated in apoptosis of Triple Negative Breast Cancer (TNBC) cells. BL EthOH was cytotoxic against TNBC cells like MDA-MB-231 and MDA-MB-453 with IC₅₀ concentrations 67.48 ± 5.45 and 70.03 ± 4.76 μg/ml, respectively. Apoptotic studies showed that BL EthOH was able to induce apoptosis and western blot studies demonstrated that BL EthOH significantly decreased the Phospho-NF-κB (ser536), PCNA, anti-apoptotic protein Bcl-2 expression and increased the expression of pro-apoptotic protein Bax, in MDA-MB-231 and MDA-MB-453 cell lines when compared with untreated cells. Besides, BL EthOH has synergistic chemosensitizing effects on TNBC cells and increased the cytotoxicity of doxorubicin and cisplatin.