Hemisynthesis and cytotoxic evaluation of manoyl oxide analogs from sclareol: effect of two tertiary hydroxyls & Heck coupling on cytotoxicity.

Sclareol, a bioactive diterpene alcohol isolated from Salvia sclarea, was subjected to structural modification and cytotoxic evaluation. Boron-Heck-coupled analogs of manoyl oxide were prepared from sclareol in a two-step reaction scheme. In the first step manoyl oxide was prepared from sclareol using cerium (IV) ammonium nitrate. Further the structural modification of manoyl oxide via Palladium (II) catalysed Boron-Heck coupling reaction produced a new series of compounds. All the synthesised compounds were screened for in vitro cytotoxic evaluation against four cancer cell lines HCT-116, MCF-7, MDA-MB231and MDA-MB468. The results showed that manoyl oxide is less active than sclareol. Sclareol shows an IC50 of 2.0 µM compared to manoyl oxide with an IC50 of 50 µM against the MCF-7 cell line. From the results it was inferred that the presence of two tertiary hydroxyls in sclareol are necessary for its cytotoxic activity and Heck coupled analogs are more active than sclareol and manoyl oxide.

Canonical DDR activation by EMT inducing agent 5-Fluorouracil is modulated by a cannabinoid based combinatorial approach via inducing autophagy and suppression of vimentin expression.

Anastasis cascade including induction of Epithelial to Mesenchymal Transition (EMT), DNA repair, and stimulation of pro-survival mediators collectively exaggerate therapy resistance in cancer prognosis. The extensive implications of DNA-damaging agents are clinically proven futile for the rapid development of disease recurrence during treatment regime. Herein we report a glycosidic derivative of Δ9-tetrahydrocannabinol (THC-9-OG) abrogates sub-toxic doses of 5-Fluorouracil (5FU) induced EMT in colon cancer cells nullifying DNA repairing mechanism. Our in vitro and in vivo data strongly proclaims that THC-9-OG could not only abrogate 5FU mediated background EMT activation through stalling matrix degradation as well as murine 4T1 lung metastasis but also vigorously diminished Rad-51 repairing mediator along with stimulation of γ-H2AX foci formation. The combinatorial treatment (5FU + THC-9-OG) in Apc knockout colorectal carcinoma model conferred remission of the crypt progenitor phenotype which was prominently identified in 5FU treatment. Mechanistically, we demonstrated that 5FU plus THC-9-OG significantly attenuated major EMT inducer Vimentin via extensive ROS generation along with autophagy induction via LC3B I-II conversion and p62 degradation in a p-ATM dependent manner. Additionally, Cannabinoid receptor CB1 was responsible for abrogation of Vimentin since we found increase in the expression of γH2AX and decrease in vimentin expression in CB1 agonist (ACEA) plus 5FU treated cells. Nutshell, our results unveil a new direction of Cannabinoid based combinatorial approach to control background EMT along with robust enhancing of DNA damage potential of sub-toxic concentration of 5FU resulting immense inhibition of distant metastasis coupled with triggering cell death in vitro and in vivo.

Divergent synthesis of fractionated Cannabis sativa extract led to multiple cannabinoids C-&O-glycosides with anti-proliferative/anti-metastatic properties.

Here, we present an interesting, previously unreported method for fractionating a particular class of cannabinoids from the crude leaf extract of Cannabis sativa using HP-20 resins. In this study, we report a novel method of divergent synthesis of fractionated Cannabis sativa extract, which allows the generation of multiple cannabinoids C- and O-glycosides which react with the glycosyl donor 2,3,4,6-tetra-O-acetyl-d-mannosyl trichloroacetimidate (TAMTA) to create eight C- and O-ß-d-cannabinoids glycosides (COCG), which are separated by HPLC and whose structures are characterized by 1D, 2D NMR, and mass spectrometry. These glycosides exhibit improved anti-proliferative and anti-metastatic effects against numerous cancer cell lines in vitro and are more water-soluble and stable than their parent cannabinoids. The in vitro testing of the pure cannabinoids (1-4) and their C- & O-glycosides (1a-4a) and 1b-4b exhibited anti-proliferative and anti-metastatic activities against a panel of eight human cancer cell lines in contrast to their respective parent molecules. Different cancer cell lines' IC50 values varied significantly when their cell viability was compared. In addition to the others, compounds 2a, 3a, 4a, and 2b, 3b were highly potent, with IC50values ranging from 0.74 µM (3a) to 51.40 µM (4a).Although2a(1.42 µM) and3a(0.74 µM) exhibited lower IC50values in the MiaPaca-2 cell line than4a(2.58 µM). But, in addition to the comparable anti-clonogenic activity of4ain MiaPaca-2 and Panc-1 cells, it manifested remarkable anti-invasive activity than either 2a or 3a.In contrast to 2a, 2b, 3a, and 3b and their respective parent compounds,4ahad substantial anti-invasive/anti-metastatic capabilities and possessed anti-proliferative activity.The effects of 4a treatment on MiaPaca-2 and Panc-1 cells include a dose-dependent increase in the expression of E-cadherin and a significant decrease in the expression of Zeb-1, Vimentin, and Snail1. Our results demonstrate that divergent synthesis of fractionated Cannabis sativa extract is a feasible and efficient strategy to produce a library of novel cannabinoid glycosides with improved pharmacological properties and potential anticancer benefits.

Synthesis and characterization of piperic acid conjugates with homochiral and heterochiral dipeptides containing phenylalanine and their application in skin cancer.

The current work demonstrates the synthesis and characterization of piperic acid conjugates with homochiral/heterochiral dipeptides containing phenylalanine as anti-skin cancer agents. The conjugates PA-DPhe-LPhe-OH, FC-1; PA-LPhe-DPhe-OH, FC-2; PA-DPhe-DPhe-OH, FC-3; and PA-DPhe-DPhe-OH, FC-4 were synthesized, characterized and assessed for cytotoxicity against melanoma cell lines of human and murine origin. Among all, PA-DPhe-DPhe-OH (FC-3) conjugate was identified as a potential cytotoxic lead against melanoma cells by delineating the anti-proliferative and anti-migratory potential together with its anti-inflammatory potential against pro-inflammatory interleukins (IL-1ß, IL-6, and IL-8). Evidences from western blotting, fractionation, and immunocytochemistry experiments suggest that Stat-3 is a critical signaling molecule involved in the FC-3 mechanism of action. The results denote that FC-3 profoundly ablates Stat-3 expression, phosphorylation, and nuclear translocation. Stat-3 mRNA analysis revealed that FC-3 did not alter the transcription of Stat-3. However, in cells where proteasome mediated degradation was inhibited, FC-3 failed to check the Stat-3 expression implying that FC-3 augments the proteasomal degradation of Stat-3. Of note, FC-3 failed to reverse the IL-6 mediated hyperactivation of Stat-3 in A375 cells. Critically, in Stat-3 deficient cancer cells, the anti-clonogenic and anti-migratory potential of FC-3 was significantly subdued. Further, the in vivo efficacy of FC-3 was validated in the two-step (DMBA/TPA) chemically induced mouse skin cancer model. The FC-3-treated cohorts of mice unveiled a significant decrease in the cumulative number of tumors besides attenuation of tumor growth with respect to the vehicle-treated mice. Lastly, in corroboration with our in vitro findings, serum collected from mice groups at various intervals during the treatment regimen demonstrated decrement in IL-1ß and IL-6 levels in FC-3 treated groups compared to the vehicle-treated group.

Diversity, Antimicrobial, Antioxidant, and Anticancer Activity of Culturable Fungal Endophyte Communities in Cordia dichotoma.

Endophytic fungi are a significant source of secondary metabolites, which are chemical compounds with biological activities. The present study emphasizes the first-time isolation and identification of such fungi and their pharmacological activities from the medicinal plant Cordia dichotoma, which is native to Jammu, India. The Shannon Wiener diversity index revealed a wide range of fungal endophytes in root (1.992), stem (1.645), and leaf (1.46) tissues. A total of 19 endophytic fungi belonging to nine different genera were isolated from this plant and the majority belonged to the Ascomycota phylum. ITS rRNA gene sequencing was used to identify the fungal strains and they were submitted in NCBI GenBank. The most potent fungal isolate Cladosporium cladosporioides OP870014 had strong antimicrobial, antioxidant, and anticancer activity against MCF-7, HCT-116, and PC-3 cancer cell lines. The LC-MS and GC-MS analyses of the ethyl acetate extract of C. cladosporioides were examined to identify the bioactive metabolites. The major compounds of the crude extract derived from C. cladosporioides OP870014, according to GC-MS, are spiculisporic acid; dibutyl phthalate; phenylethyl alcohol; cyclohexanone, 2,3,3-trimethyl-2-3-methylbutyl; pyrrolo[1,2-a]pyrazine-1,4-dione,hexahydro-3-(phenylmethyl);2,5-piperazinedione,3,6-bis(2-methylpropyl); and heneicosane which possessed antimicrobial, anticancerous, and antioxidant activities. The findings revealed that C. dichotoma has the capacity to host a wide variety of fungal endophytes and that secondary metabolites from the endophytic fungus may be a source of alternative naturally occurring antimicrobial, antioxidant, and cytotoxic compounds.

Quinoxalinone substituted pyrrolizine (4h)-induced dual inhibition of AKT and ERK instigates apoptosis in breast and colorectal cancer by modulating mitochondrial membrane potential.

AKT and ERK 1/2 play a pivotal role in cancer cell survival, proliferation, migration, and angiogenesis. Therefore, AKT and ERK 1/2 are considered crucial targets for cancer intervention. In this study, we envisaged the role of AKT and ERK signaling in apoptosis regulation in presence of compound 4h, a novel synthetic derivative of quinoxalinone substituted spiropyrrolizines exhibiting substantial antiproliferative activity in various cancer cell lines. Structurally 4h is a spiropyrrolizine derivative. Molecular docking analysis revealed that compound 4h shows strong binding affinity with AKT-1 (-9.5 kcal/mol) and ERK2 (-9.0 kcal/mol) via binding at allosteric sites of AKT and active site of ERK2. The implications of 4h binding with these two survival kinases resulted in the obstruction for ATP binding, hence, hampering their phosphorylation dependent activation. We demonstrate that 4h mediated apoptotic induction via disruption in the mitochondrial membrane potential of MCF-7 and HCT-116 cells and 4h-mediated inhibition of survival pathways occurred in a wild type PTEN background and is diminished in PTEN-/- cells. In 4T1 mammary carcinoma model, 4h exhibited pronounced reduction in the tumor size and tumor volume at significantly low doses. Besides, 4h reached the highest plasma concentration of 5.8 µM within a period of 1 h in mice model intraperitoneally. Furthermore, 4h showed acceptable clearance with an adequate elimination half-life and satisfactory pharmacokinetic behaviour, thus proclaiming as a potential lead molecule against breast and colorectal cancer by specifically inhibiting simultaneously AKT and ERK1/2 kinases.

Identification of new modulator of DNA repairing pathways based on natural product (±)-peharmaline A.

The complex heterogenic environment of tumour mass often leads to drug resistance and facilitate chemo insensitivity triggering more malignant phenotypes among cancer patients. Major DNA-damaging cancer drugs have been consistently proven unsuccessful in terms of elevating chemo-resistance. (±)-peharmaline A, a hybrid natural product isolated from seeds of Peganum harmala L. possesses significant cytotoxic activities. Herein, we have described the design, and synthesis of a novel library of close and simplified analogues around the anticancer natural product (±)-peharmaline A and investigated their cytotoxic activities, which led to the identification of three structurally simplified lead compounds exhibiting better potency than parent natural product. Among them, demethoxy analogue of peharmaline A was further investigated for its anticancer potential eliciting demethoxy analogue as potent DNA-damage inducing agent attenuating the expression of the proteins responsible for the DNA damage repair. Therefore, this demethoxy analogue warrants detailed investigations for the confirmations of the molecular mechanism-based studies responsible for its anticancer activity. ______________________________________________________________________________.

Glabridin plays dual action to intensify anti-metastatic potential of paclitaxel via impeding CYP2C8 in liver and CYP2J2/EETs in tumor of an orthotopic mouse model of breast cancer.

In spite of unprecedented advances in modern cancer therapy, there is still a dearth of targeted therapy to circumvent triple-negative breast cancer (TNBC). Paclitaxel is the front-line therapy against TNBC, but the main constraints of its treatment are dose-related adverse effects and emerging chemoresistance. In this context, glabridin (phytoconstituent from Glycyrrhiza glabra) is reported to hit multiple signalling pathways at the in-vitro level, but hardly any information is known at the in-vivo level. We aimed here to elucidate glabridin potential with an underlying mechanism in combination with a low dose of paclitaxel using a highly aggressive mouse mammary carcinoma model. Glabridin potentiated the anti-metastatic efficacy of paclitaxel by substantially curtailing tumor burden and diminishing lung nodule formation. Moreover, glabridin remarkably attenuated epithelial-mesenchymal transition (EMT) traits of hostile cancer cells via up-regulating (E-cadherin & occludin) and down-regulating (Vimentin & Zeb1) vital EMT markers. Besides, glabridin amplified apoptotic induction effect of paclitaxel in tumor tissue by declining or elevating pro-apoptotic (Procaspase-9 or Cleaved Caspase-9 & Bax) and reducing anti-apoptotic (Bcl-2) markers. Additionally, concomitant treatment of glabridin and paclitaxel predominantly lessened CYP2J2 expression with marked lowering of epoxyeicosatrienoic acid (EET)'s levels in tumor tissue to reinforce the anti-tumor impact. Simultaneous administration of glabridin with paclitaxel notably enhanced plasma exposure and delayed clearance of paclitaxel, which was mainly arbitrated by CYP2C8-mediated slowdown of paclitaxel metabolism in the liver. The fact of intense CYP2C8 inhibitory action of glabridin was also ascertained using human liver microsomes. Concisely, glabridin plays a dual role in boosting anti-metastatic activity by augmenting paclitaxel exposure via CYP2C8 inhibition-mediated delaying paclitaxel metabolism and limiting tumorigenesis via CYP2J2 inhibition-mediated restricting EETs level. Considering the safety, reported protective efficacy, and the current study results of boosted anti-metastatic effects, further investigations are warranted as a promising neoadjuvant therapy for crux paclitaxel chemoresistance and cancer recurrence.

Rottlerin renders a selective and highly potent CYP2C8 inhibition to impede EET formation for implication in cancer therapy.

CYP2C8 is a crucial CYP isoform responsible for the metabolism of xenobiotics and endogenous molecules. CYP2C8 converts arachidonic acid to epoxyeicosatrienoic acids (EETs) that cause cancer progression. Rottlerin possess significant anticancer actions. However, information on its CYP inhibitory action is lacking in the literature and therefore, we aimed to explore the same using in silico, in vitro, and in vivo approaches. Rottlerin showed highly potent and selective CYP2C8 inhibition (IC50 < 0.1 µM) compared to negligible inhibition (IC50 > 10 µM) for seven other experimental CYPs in human liver microsomes (HLM) (in vitro) using USFDA recommended index reactions. Mechanistic studies reveal that rottlerin could reversibly (mixed-type) block CYP2C8. Molecular docking (in silico) results indicate a strong interaction could occur between rottlerin and the active site of human CYP2C8. Rottlerin boosted the plasma exposure of repaglinide and paclitaxel (CYP2C8 substrates) by delaying their metabolism using the rat model (in vivo). Multiple-dose treatment of rottlerin with CYP2C8 substrates lowered the CYP2C8 protein expression and up-regulated & down-regulated the mRNA for CYP2C12 & CYP2C11 (rat homologs), respectively, in rat liver tissue. Rottlerin substantially hindered the EET formation in HLM. Overall results of rottlerin on CYP2C8 inhibition and EET formation insinuate further exploration for cancer therapy.

ß-(4-fluorobenzyl) Arteannuin B induced interaction of ATF-4 and C/EBPß mediates the transition of breast cancer cells from autophagy to senescence.

ATF-4 is a master regulator of transcription of genes essential for cellular-adaptive function. In response to the quantum and duration of stress, ATF-4 diligently responds to both pro-apoptotic and pro-survival signals converging into either autophagy or apoptosis/senescence. Despite emerging cues implying a relationship between autophagy and senescence, how these two processes are controlled remains unknown. Herein, we demonstrate ß-(4-fluorobenzyl) Arteannuin B (here after Arteannuin 09), a novel semisynthetic derivative of Arteannuin B, as a potent ER stress inducer leading to the consistent activation of ATF-4. Persistent ATF-4 expression at early time-points facilitates the autophagy program and consequently by upregulating p21 at later time-points, the signaling is shifted towards G2/M cell cycle arrest. As bZIP transcription factors including ATF-4 are obligate dimers, and because ATF-4 homodimers are not highly stable, we hypothesized that ATF-4 may induce p21 expression by physically interacting with another bZIP family member i.e., C/EBPß. Our co-immunoprecipitation and co-localization studies demonstrated that ATF-4 is principally responsible for the autophagic potential of Arteannuin 09, while as, induction of both ATF-4 and C/EBPß is indispensable for the p21 regulated-cell cycle arrest. Interestingly, inhibition of autophagy signaling switches the fate of Arteannuin 09 treated cells from senescence to apoptosis. Lastly, our data accomplished that Arteannuin 09 is a potent inhibitor of tumor growth and inducer of premature senescence in vivo.

Synthesis, conformation and cytotoxic activity of short hybrid peptides containing conformationally constrained 1-(aminomethyl)cyclohexanecarboxylic acid and gabapentin.

The present work describes the synthesis,conformation and cytotoxic activities of short ß/γ hybrid peptides, Boc-ß2,2-Ac6c-Gpn-NHMe, BG1; Boc-(ß2,2-Ac6c-Gpn)2-OMe, BG2; Boc-(ß2,2-Ac6c-Gpn)3-OMe, BG3; H-ß2,2-Ac6c-Gpn-NHMe, BG4; H-(ß2,2-Ac6c-Gpn)2-OMe, BG5; H-(ß2,2-Ac6c-Gpn)3-OMe, BG6, Boc-ß2,2-Ac6c-Gpn-OMe, BG7 and H-ß2,2-Ac6c-Gpn-OMe, BG8. Mixed C6/C7 conformations were observed for ß/γ hybrid peptides. Further, BG1-BG8 were screened against MCF-7 (Breast cancer), A549 (Lung Cancer), PC-3 (Prostate cancer), HCT-116 (Colon cancer), and MDA-MB-231 (Breast cancer) cell lines. Among all, BG6 exhibited potent cytotoxicity against all cancer cell lines with IC50 ranging from 1.6 µM to 6.3 µM with relatively low cytotoxicity against normal epithelial breast cell line fR-2 and human embryonic kidney cell line HEK-293. Minimal hemolytic activity was observed for BG6 against human erythrocytes. Peptide BG6 displayed anti-migratory and anti-invasive potentials showing strong interactions with intrinsic apoptotic markers Bcl-2, Bax, and cleaved-PARP, as well as the induction of the mitochondria maladjustment mediated apoptosis.

Heterochiral dipeptide d-phenylalanyl- l-phenylalanine (H-D Phe-L Phe-OH) as a potential inducer of metastatic suppressor NM23H1 in p53 wild-type and mutant cells.

In recent years, significant progress has been made to the use-case of small peptides because of their diversified edifice and hence their versatile application scope in cancer therapy. Here we identify the heterochiral dipeptide H-D Phe-L Phe-OH (F1) as a potent inducer of the metastatic suppressor NM23H1. We divulge the effect of F1 on the major EMT/metastasis-associated genes and the implications on the invasion and migration ability of cancer cells. The anti-invasive potential of F1 was directly correlated with NM23H1 expression. Mechanistically, F1 treatment elevated p53 levels as validated by localization and transcriptional studies. In the NM23H1 knockdown condition, F1 failed to induce any p53 expression/nuclear localization, indicating that the upregulation in p53 expression by F1 is NM23H1 dependent. We also demonstrate how the antimetastatic potential of F1 is primarily mediated through NM23H1 irrespective of the p53 status of the cell. However, both NM23H1 and a functional p53 protein in conjunction govern the apoptotic and cytostatic potential of F1. Coimmunoprecipitation studies unraveled the augmentation of the p53 and NM23H1 interaction in p53 wild-type cells. However, in p53 mutated cells, no such enrichment was evidenced. We employed mouse isogenic cell lines (4T-1 and 4T-1 p53) to determine the in vivo efficacy of F1 (spontaneous and experimental models). Decreased tumor volume in the cohort injected with 4T-1 p53 cells demonstrated that while the antimetastatic potential of F1 was reliant on NM23H1, p53 activation was required for ablation of primary tumor burden. Our findings unravel that F1 treatment induces significant abrogation of the migration, invasion and metastatic potential of both p53 wild-type and p53 deficient cancers mediated through NM23H1.

Rottlerin promotes anti-metastatic events by ameliorating pharmacological parameters of paclitaxel: An in-vivo investigation in the orthotopic mouse model of breast cancer.

Despite substantial breakthroughs in cancer research, there is hardly any specific therapy available to date that can alleviate triple-negative breast cancer (TNBC). Paclitaxel is the first-line chemotherapy option, but its treatment is often associated with early discontinuation of therapy due to the development of resistance and/or precipitation of severe side effects. In the quest to establish a suitable combination therapy with a low dose of paclitaxel, we explored rottlerin (a pure and characterized phytoconstituent from Mallotus philippensis) because of its multifaceted pharmacological actions against cancer. The study was performed to assess the therapeutic effects of rottlerin (5-20 mg/kg) with a low dose of paclitaxel (5 mg/kg) using a highly aggressive mouse mammary carcinoma model. Rottlerin augmented the paclitaxel effect by reducing tumor burden as well as metastatic lung nodules formation. Rottlerin in combination with paclitaxel remarkably altered the expression of vital epithelial-mesenchymal transition (EMT) markers such as E-cadherin, Snail 1, & Vimentin and thus improved the anti-metastatic efficacy of paclitaxel. Significant attenuation of anti-apoptotic protein (Bcl-2) along with amplification of pro-apoptotic (cleaved PARP) marker confers that rottlerin could ameliorate the pro-apoptotic potential of paclitaxel. In this study, a rational combination of rottlerin and paclitaxel treatment curtailed CYP2J2 expression and epoxyeicosatrienoic acids (EETs) levels, responsible for restrain tumor growth and metastasis. Additionally, rottlerin lessened paclitaxel treatment-mediated hematological alterations and prevented paclitaxel treatment-linked key serum biochemical changes related to organ toxicities. These rottlerin treatment-mediated protective changes are closely associated with the lower paclitaxel accumulation in the corresponding tissues. Rottlerin caused significant pharmacokinetic interaction with paclitaxel to boost the plasma level of paclitaxel in a typical mouse model and possibly helpful towards the use of a low dose of paclitaxel in combination. Overall, it can be stated that rottlerin has significant potential to augment the anti-metastatic efficacy of paclitaxel via impeding EMT activation along with attenuating its treatment-associated toxicological alterations. Hence, rottlerin has significant potential to explore further as a suitable neoadjuvant therapy with paclitaxel against TNBC.

Isolation and anticancer activity evaluation of rare Bisaryl anthraquinone antibiotics from novel Streptomyces sp. strain of NW Himalayan region.

Biosynthesis of bisaryl preanthraquinone antibiotics by various microorganisms differs in monomeric subunits as well as their dimerization positions leading to different configurations. The present study relates to the production of rare bisaryl anthraquinone antibiotics by a new Streptomyces strain isolated from Shivalik region of NW Himalayas. In vitro anticancer and anti-migratory effects of Setomimycin (9,9' bisanthraquinone antibiotic) was seen with a significant reduction in the expression of both MEK as well as ERK pathways in a dose dependent manner at 6.5 µM & 8 µM concentration in HCT-116 and 5.5 µM & 7 µM concentration in MCF-7 cells. In vivo studies in aggressive orthotopic mouse mammary carcinoma model (4T1) demonstrated about 76% reduction of primary tumor weight and 90.5% reduction in the tumor volume within two weeks. In vivo pharmacokinetics study of setomimycin revealed that it can be rapidly absorbed with an adequate plasma exposure and half-life which can be linked to its in vivo efficacy.

Palladium catalyzed migratory heck coupling of arteannuin B and boronic acids: An approach towards the synthesis of antiproliferative agents in breast and lung cancer cells.

We have recently highlighting the role of spiroisoxazoline arteannuin B derivatives in mediating proinflammatory cytokines like IL-6, TNfα and NO in vitro. In the present study, a series of new ß-arylated arteannuin B analogues were synthesized through coupling with arylboroic acids and evaluated for their in vitro cytotoxic activity in a panel of six cancer cell lines. The binding efficiency was verified by docking of the original ligand within the active site of ATPase domain of GRP78 (PDB ID: 3LDL) at a resolution of 2.30 Å with the score energy of -8.07 kcal/mol. Among the new compounds 3a, 3b, 3d, 3i, 3j and 3n displayed potent cytotoxic potential with an IC50 from 2 to 18 µM and compound 3i was proven to be the most potent cytotoxic and anti-proliferative compound of all the six distinct cell lines. Compound 3i exhibited promising apoptosis inducing potential in breast cancer cells and stalled their wound healing properties and was effective in blocking the migration of cancer cells.

Natural podophyllotoxin analog 4DPG attenuates EMT and colorectal cancer progression via activation of checkpoint kinase 2.

Epithelial-mesenchymal transition (EMT) is critical for the metastatic dissemination of cancer cells and contributes to drug resistance. In this study, we observed that epithelial colorectal cancer (CRC) cells transiently exposed to 5-fluorouracil (5-FU) (a chemotherapeutic drug for CRC) as well as 5-FU-resistant cells (5-FU-R) develop EMT characters as evidenced by activation of Vimentin and augmented invasive properties. On the other hand, 4DPG (4'-demethyl-deoxypodophyllotoxin glucoside), a natural podophyllotoxin analog attenuates EMT and invadopodia formation abilities of HCT-116/5-FU-R and SW-620/5-FU-R cells. Treatment with 4DPG restrains Vimentin phosphorylation (Ser38) in 5-FU-R cells, along with downregulation of mesenchymal markers Twist1 and MMP-2 while augmenting the expression of epithelial markers E-cadherin and TIMP-1. Moreover, 4DPG boosts the tumor-suppressor protein, checkpoint kinase 2 (Chk2) via phosphorylation at Thr68 in a dose-dependent manner in 5-FU-R cells. Mechanistically, SiRNA-mediated silencing of Chk2, as well as treatment with Chk2-specific small-molecule inhibitor (PV1019), divulges that 4DPG represses Vimentin activation in a Chk2-dependent manner. Furthermore, immunoprecipitation analysis unveiled that 4DPG prevents complex formation between Vimentin and p53 resulting in the rescue of p53 and its nuclear localization in aggressive 5-FU-R cells. In addition, 4DPG confers suitable pharmacokinetic properties and strongly abrogates tumor growth, polyps formation, and lung metastasis in an orthotopic rat colorectal carcinoma model. In conclusion, our findings demonstrate 4DPG as a targeted antitumor/anti-metastatic pharmacological lead compound to circumvent EMT-associated drug resistance and suggest its clinical benefits for the treatment of aggressive cancers.

Benzimidazole-Based Organic-Inorganic Gold Nanohybrids Suppress Invasiveness of Cancer Cells by Modulating EMT Signaling Cascade.

Over the past few years, nanotechnology-based approaches have emerged to override drug resistance owing to their superiority over other formulations because of their diverse therapeutic advantages such as target-specific drug delivery, enhanced bioavailability, biodegradability, and minimal off-target effects. Hybrid nanomaterials as a formulation of anticancer drugs with gold nanoparticles (AuNPs) have adequately proven efficacious in controlled release as well as disintegration into ultrasmall nanoparticles dragging the drug to penetrate deep into tumor tissues and consequently getting cleared from the body. In this study, to achieve better antitumor responses, we engineered self-assembled organic nanoparticles of potent anticancer compound BZ6 (BZ6-ONPs), BZ6-gold nanoparticle conjugates (BZ6-AuNPs), and organic-inorganic nanohybrids involving amalgamation of AuNPs with BZ6-ONPs (AuNPs@BZ6-ONPs) and comparatively analyzed their physicochemical as well as biological activities. The epithelial-mesenchymal transition (EMT) is a critical biological event that facilitates metastatic spread of cancer cells and contributes to chemoresistance. AuNPs@BZ6-ONPs consistently suppressed EMT characteristics including invasion, cell scattering, and migration abilities of aggressive breast cancer (MDA-MB-231) and pancreatic adenocarcinoma (PANC-1) cells much more efficiently than BZ6-ONPs and BZ6-AuNPs. Western blotting and immunocytochemistry analysis unveiled that the nanohybrids downregulated expression of the key mesenchymal markers NF-κß p65, Twist-1, vimentin, and MMP-2, meanwhile augmenting epithelial marker E-cadherin and tumor suppressor Par-4. The in vivo syngenic mouse tumor model demonstrated remarkable reduction of tumor growth (84.3%) and metastatic lung nodules (66.1%) following 14 days of treatment without any adverse effects. Finally, the facile and ecofriendly method of synthesis of AuNPs@BZ6-ONPs demonstrating promising antitumor/antimetastatic efficacies suggests its therapeutic implication for the treatment of advanced cancers.

Overlapping targets exist between the Par-4 and miR-200c axis which regulate EMT and proliferation of pancreatic cancer cells.

The last decade has witnessed a substantial expansion in the field of microRNA (miRNA) biology, providing crucial insights into the role of miRNAs in disease pathology, predominantly in cancer progression and its metastatic spread. The discovery of tumor-suppressing miRNAs represents a potential approach for developing novel therapeutics. In this context, through miRNA microarray analysis, we examined the consequences of Prostate apoptosis response-4 (Par-4), a well-established tumor-suppressor, stimulation on expression of different miRNAs in Panc-1 cells. The results strikingly indicated elevated miR-200c levels in these cells upon Par-4 overexpression. Intriguingly, the Reverse Phase Protein Array (RPPA) analysis revealed differentially expressed proteins (DEPs), which overlap between miR200c- and Par-4-transfected cells, highlighting the cross-talks between these pathways. Notably, Phospho-p44/42 MAPK; Bim; Bcl-xL; Rb Phospho-Ser807, Ser811; Akt Phospho-Ser473; Smad1/5 Phospho-Ser463/Ser465 and Zyxin scored the most significant DEPs among the two data sets. Furthermore, the GFP-Par-4-transfected cells depicted an impeded expression of critical mesenchymal markers viz. TGF-ß1, TGF-ß2, ZEB-1, and Twist-1, concomitant with augmented miR-200c and E-cadherin levels. Strikingly, while Par-4 overexpression halted ZEB-1 at the transcriptional level; contrarily, silencing of endogenous Par-4 by siRNA robustly augmented the Epithelial-mesenchymal transition (EMT) markers, along with declining miR-200c levels. The pharmacological Par-4-inducer, NGD16, triggered Par-4 expression which corresponded with increased miR-200c resulting in the ZEB-1 downregulation. Noteworthily, tumor samples obtained from the syngenic mouse pancreatic cancer model revealed elevated miR-200c levels in the NGD16-treated mice that positively correlated with the Par-4 and E-cadherin levels in vivo; while a negative correlation was evident with ZEB-1 and Vimentin.

Par-4 activation restrains EMT-induced chemoresistance in PDAC by attenuating MDM-2.

BACKGROUND: We recently reported prostate apoptosis response 4 (Par-4), a potential tumor suppressor protein restrains epithelial-mesenchymal transition (EMT) properties and promotes mesenchymal-epithelial transition (MET) in invasive cancer cells by repressing Twist-1 promoter activity. Here, we demonstrate that genetic as well as pharmacological modulation of Par-4 by NGD16 (a small molecule antimetastatic agent), limits EMT-induced chemoresistance in aggressive cancer cells by suppressing MDM-2, a downstream effector of Twist-1. METHODS: Matrigel invasion assay, gelatin degradation assay, cell scattering assay, MTT assay and colony formation assay were used to study the proliferation and migration abilities of invasive cancer cells. Immunoblotting, immunocytochemistry, and immunoprecipitation analysis were utilized for determining protein expression and protein-protein interaction. 4T1 aggressive mouse carcinoma model was employed to evaluate tumor growth and lung metastasis. RESULTS: Treatment of gemcitabine (nucleoside analogue anticancer agent) to pancreatic cancer (Panc-1, MiaPaca-2) and breast cancer (MDA-MB-231) cells amplified MDM-2 expression along with increase in EMT properties. Conversely, NGD16 boosted expression of tumor suppressor Par-4 and inhibited invasion and migration abilities of these cells. Moreover, induction of Par-4 effectively diminished MDM-2 along with pro-EMT markers, whereas, augmented the expression of epithelial markers. Furthermore, siRNA-mediated silencing of Par-4 divulged that NGD16 exerts its EMT inhibitory effects in a Par-4-dependent manner. Mechanistically, Par-4 activation provokes p53 by disrupting MDM-2-p53 interaction, which restored epithelial characteristics in cancer cells. Additionally, partial knockdown of MDM-2 through siRNA pronounced the anti-proliferative and anti-invasive effects of NGD16. Finally, NGD16 efficiently inhibited tumor growth and lung metastasis in mouse mammary carcinoma model without showing any undesirable effects. CONCLUSION: Our findings unveil Par-4 as a key therapeutic target and NGD16 (the pharmacological modulator of Par-4) are potential tools to suppress EMT and associated chemoresistance, which could be exploited clinically for the treatment of aggressive cancers.