Browsing by Author "Tripathi, Amit K."
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Item A Drug-Loaded Nanoparticle to Target Bone-Metastatic Prostate Cancer(2022) Lampe, Jana B.; Desai, Priyanka; Tripathi, Amit K.; Ranjan, Amalendu; Vishwanatha, JamboorTreatment for localized prostate cancer (PCa) has a tremendous success rate. However, the fact that the five-year overall survival rate drops from 100% to 30.2% when tumor cells metastasize to distant sites, represents an unmet medical need. In 90% of metastatic cases, bone is the primary metastatic site. Our objective is to co-load a poly(lactic-co-glycolic) (PLGA) nanoparticle (NP) with Cabazitaxel (CBZ) and Bortezomib (BTZ) and to conjugate a bone-targeting moiety, Alendronate (ALN), to the outside of the nanoparticle to facilitate targeting to bone tumors and to ameliorate the resulting bone damage. We hypothesize that this targeted nanomedicine will affect genes and proteins that contribute to invasion and migration, anti-apoptotic signaling, and ultimately lead to tumor-cell apoptosis. Furthermore, we predict that the nano-delivery system will help ameliorate bone lesions inflicted by the tumors. Methods: Nanoparticles were engineered using an Emulsion-Diffusion-Evaporation Technique in which PLGA is dissolved in dichloromethane, 5% polyvinyl alcohol, and Bis(sulfosuccinimydyl)suberate (BS3) crosslinker. For targeting, Alendronate (ALN) is later conjugated to the outside of the nanoparticle via this crosslinker. Results: Our average NP size was around 240 nm in diameter, a PDI of < 0.2, with a Zeta Potential (ZP) of -28 mV. Our drug loading capacity (DL) for CBZ was 11.97% and for BTZ 0.9%. Encapsulation efficiency (EE) for CBZ was 25.26% and 8.9% for BTZ. The IC50 for the CBZ NPs is 5.6 nM and BTZ NPs is 15.6 nM. We have successfully shown that the gene expression for various migration and invasion markers as well as cell signaling proteins have been affected by the nanoparticles. Conclusions: Our nanoparticles have a desirable size, PDI, ZP, DL, and EE for our intended therapeutic purpose. Furthermore, we have shown alterations in the cell signaling and gene expression responsible for Epithelial-to-Mesenchymal Transition-Transcription Factors (EMT-TFs), indicating that our nanotherapeutic has significant potential to treat metastatic PCa and to mitigate the damage done by metastatic tumors.Item Cabazitaxel-Loaded Nanoparticles Reduce the Invasiveness in Metastatic Prostate Cancer Cells: Beyond the Classical Taxane Function(MDPI, 2023-02-26) Lampe, Jana B.; Desai, Priyanka P.; Tripathi, Amit K.; Sabnis, Nirupama A.; Chen, Zhe; Ranjan, Amalendu P.; Vishwanatha, Jamboor K.Bone-metastatic prostate cancer symbolizes the beginning of the later stages of the disease. We designed a cabazitaxel-loaded, poly (lactic-co-glycolic acid) (PLGA) nanoparticle using an emulsion-diffusion-evaporation technique. Bis (sulfosuccinimidyl) suberate (BS3) was non-covalently inserted into the nanoparticle as a linker for the conjugation of a bone-targeting moiety to the outside of the nanoparticle. We hypothesized that the nanoparticles would have the ability to inhibit the epithelial-to-mesenchymal transition (EMT), invasion, and migration in prostate cancer cells. Targeted, cabazitaxel-loaded nanoparticles attenuated the EMT marker, Vimentin, and led to an increased E-cadherin expression. These changes impart epithelial characteristics and inhibit invasive properties in cancer progression. Consequently, progression to distant sites is also mitigated. We observed the reduction of phosphorylated Src at tyrosine 416, along with increased expression of phosphorylated cofilin at serine 3. These changes could affect migration and invasion pathways in cancer cells. Both increased p-120 catenin and inhibition in IL-8 expression were seen in targeted, cabazitaxel-loaded nanoparticles. Overall, our data show that the targeted, cabazitaxel-loaded nanoparticles can act as a promising treatment for metastatic prostate cancer by inhibiting EMT, invasion, and migration, in prostate cancer cells.Item Combination of Small Extracellular Vesicle-Derived Annexin A2 Protein and mRNA as a Potential Predictive Biomarker for Chemotherapy Responsiveness in Aggressive Triple-Negative Breast Cancer(MDPI, 2023-01-09) Desai, Priyanka P.; Narra, Kalyani; James, Johanna D.; Jones, Harlan P.; Tripathi, Amit K.; Vishwanatha, Jamboor K.Small extracellular vesicles (sEVs), mainly exosomes, are nanovesicles that shed from the membrane as intraluminal vesicles of the multivesicular bodies, serve as vehicles that carry cargo influential in modulating the tumor microenvironment for the multi-step process of cancer metastasis. Annexin A2 (AnxA2), a calcium(Ca(2+))-dependent phospholipid-binding protein, is among sEV cargoes. sEV-derived AnxA2 (sEV-AnxA2) protein is involved in the process of metastasis in triple-negative breast cancer (TNBC). The objective of the current study is to determine whether sEV-AnxA2 protein and/or mRNA could be a useful biomarkers to predict the responsiveness of chemotherapy in TNBC. Removal of Immunoglobulin G (IgG) from the serum as well as using the System Bioscience's ExoQuick Ultra kit resulted in efficient sEV isolation and detection of sEV-AnxA2 protein and mRNA compared to the ultracentrifugation method. The standardized method was applied to the twenty TNBC patient sera for sEV isolation. High levels of sEV-AnxA2 protein and/or mRNA were associated with stage 3 and above in TNBC. Four patients who responded to neoadjuvant chemotherapy had high expression of AnxA2 protein and/or mRNA in sEVs, while other four who did not respond to chemotherapy had low levels of AnxA2 protein and mRNA in sEVs. Our data suggest that the sEV-AnxA2 protein and mRNA could be a combined predictive biomarker for responsiveness to chemotherapy in aggressive TNBC.Item Higher Expression of Annexin A2 in Metastatic Bladder Urothelial Carcinoma Promotes Migration and Invasion(MDPI, 2022-11-27) Guo, Christina; Trivedi, Rucha; Tripathi, Amit K.; Nandy, Rajesh; Wagner, Diana C.; Narra, Kalyani; Chaudhary, PankajIn this study, we aim to evaluate the significance of AnxA2 in BLCA and establish its metastatic role in bladder cancer cells. Analysis of TCGA data showed that AnxA2 mRNA expression was significantly higher in BLCA tumors than in normal bladder tissues. High mRNA expression of AnxA2 in BLCA was significantly associated with high pathological grades and stages, non-papillary tumor histology, and poor overall survival (OS), progression-free survival (PFS), and diseases specific survival (DSS). Similarly, we found that AnxA2 expression was higher in bladder cancer cells derived from high-grade metastatic carcinoma than in cells derived from low-grade urothelial carcinoma. AnxA2 expression significantly mobilized to the surface of highly metastatic bladder cancer cells compared to cells derived from low-grade tumors and associated with high plasmin generation and AnxA2 secretion. In addition, the downregulation of AnxA2 cells significantly inhibited the proliferation, migration, and invasion in bladder cancer along with the reduction in proangiogenic factors and cytokines such as PDGF-BB, ANGPT1, ANGPT2, Tie-2, bFGF, GRO, IL-6, IL-8, and MMP-9. These findings suggest that AnxA2 could be a promising biomarker and therapeutic target for high-grade BLCA.Item Looking Beyond Standard Chemotherapy: Peptides in Breast Cancer Treatment(2023) Tripathi, Amit K.; Vishwanatha, Jamboor K.Background: Chemotherapy is the most established method of treatment that kill fast-dividing cancer cells. However, most cancer drugs have very poor cell selectivity and kill normal cells along with cancer cells indiscriminately. Besides, the continuous use of this therapy increases the possibility of drug resistance in the body along with the chances of recurrence. The usage of peptide-based drugs to combat cancer is gaining significance in the pharmaceutical industry. The collateral damage caused to normal cells due to the use of chemotherapy, radiotherapy, etc. has given an impetus to the search for alternative methods of cancer treatment. Candidate Protein: In the fight against cancer, newer strategies to combat cancer progression are of utmost importance. Peptides derived from naturally occurring proteins are an important strategy to identify lead molecules in the field of cancer therapeutics. Migration and invasion enhancer 1 (MIEN1) is one such candidate protein that is overexpressed in various cancers and plays an important role in cancer cell migration and invasion. Conserved regions of ITAM and prenylation motif in MIEN1 were used as a template to identify anti-cancer peptides. In vitro Results: The two newly identified bioactive peptides (named LA3IK and RP-7) inhibited genes and proteins responsible for cancer cell migration and invasion in both MDA-MB-231 breast cancer. RNA-seq, qPCR analysis and western blots showed changes in the transcriptome and protein expression after peptide treatment. The mechanism of the action of the peptides involves the inhibition of key pathways like Epithelial-Mesenchymal transition and Epidermal Growth Factor-mediated NF-κB pathway to exert their anti-cancer activity. Interestingly, the peptides targeted the same signal transduction pathways followed by parental MIEN1 to show their anti-cancer properties. Thus, the two peptides acted as dominant negative effectors of MIEN1 activity. In vivo results: Additionally, LA3IK and RP-7 peptide treatments induced apoptosis in mice groups bearing tumors derived from MDA-MB-231 cells as evidenced by increased levels of cleaved caspase-3 and PARP proteins in western blots. Intriguingly, the MIEN1 mRNA and protein levels were lowered in the in vivo breast cancer tumor models that remained unchanged in in vitro experiments indicating an improved therapeutic activity in the living system. The peptides did not cause any toxicity in the mice group that received peptides only, at three times the dose used during in vivo assays. Pharmacokinetic studies: The PK studies along with the half-life determination and plasma-binding studies are underway in collaboration with the Preclinical Pharmacology Core of UT Southwestern Medical Center to identify and improve the drug-like characteristics of the MIEN1-protein derived anti-cancer peptides LA3IK and RP-7. IP Status: An intellectual property right application has been filed for LA3IK and RP-7 in August 2022. The patent status is pending. Acknowledgments: The work was supported by The Texas Center for Health Disparities (TCHD) under the award number: U54MD006882Item MIEN1 derived peptides exhibit anti-cancer activities by inhibiting the key pathways viz. epithelial-mesenchymal transition (EMT) and NF-kB signaling in MDA-MB-231 breast Cancer Cells(2022) Tripathi, Amit K.; Desai, Priyanka; Vishwanatha, JamboorPurpose MIEN1 is a tumor-specific target protein that promotes cancer cell migration and invasion. MIEN1 is overexpressed in human breast, prostate, colorectal, gastric, ovarian, squamous cell carcinoma and non-small cell lung cancer (NSCLC) with minimal or no expression in normal cells, which makes it an excellent therapeutic target The overall goal of this project is to identify small inhibitory or interference peptides (iPeps) from the native MIEN1 protein, which could achieve the high selectivity for the native MIEN1 protein as well as being able to enter cells and inhibit the intracellular targets Methods Online tools like CASTp server were used to identify pockets or empty concavities on the MIEN1 protein surface into which solvent and other potential inhibitory molecules can gain access. Peptides were designed from the primary sequence of MIEN1 protein using various biophysical parameters and peptide designing tools such as AntiCp and Cancer PPD. Chimera program was used to check if these peptides can bind to MIEN1 protein. The peptides were synthesized and correct molecular weights were ascertained. Biological experiments like MTT and scratch assays were performed. RT-PCR was done to check the ability of the small inhibitory or interference peptides (iPeps) to inhibit the key pathways like the epithelial-mesenchymal transition (EMT) by which epithelial cells lose their cell polarity and cell-cell adhesion, and gain migratory and invasive properties to become mesenchymal stem cells. Western blot experiments were done to study the inhibition of the NFκB signaling pathway which is involved extensively in cancer development and progression. To show the specificity of these peptides for MIEN1 protein, circular dichroism (CD) experiments were performed. Results Two peptides designed based on MIEN1 protein sequence showed inhibition of MDA-MB-231 breast cancer cell proliferation in the MTT assay. Wound healing and transwell invasion assays show that these peptides inhibited the migration and invasion of MDA-MB-231 cells in a dose-dependent manner. RT-PCR results showed that the key molecules of epithelial-mesenchymal transition (EMT) were inhibited which can inhibit the migratory capacities of the cells. The nuclear translocation of p50 and p65 subunits of NFκB were also inhibited. The bio-active peptides attained a β-sheet structure in MIEN1 protein environment, in the circular dichroism (CD) experiments indicating the specific interaction with MIEN1 protein while the non-active peptides remained random coiled. Conclusions This work is the first report of the inhibitors designed for targeting MIEN1 protein. The peptides inhibitors were able to inhibit the key epithelial-to-mesenchymal transition transcription factors (EMT-TFs) like SNAIL, SLUG and TWIST1 which play a vital role in the metastatic process of breast cancer. Besides this the also downregulated nuclear factor-κB (NF-κB) activation. By inhibiting these two key processes aggressiveness and metastatic potential of MDA-MB-231 breast cancer cells were decreased.Item Phosphorylated Annexin A2 at Tyrosine 23 Regulates Exosome Release and Biogenesis in Triple Negative Breast Cancer(2022) Desai, Priyanka P.; Tripathi, Amit K.; Donkor, Michael; Thyagarajan, Srikantha; Jones, Harlan; Van Treuren, Timothy; Lampe, Jana B.; Chaudhary, Panka J.; Vishwanatha, JamboorPurpose: Exosomes are highly involved in the progression of diverse diseases. Targeting exosome biogenesis and release is a potential strategy for the treatment of the disease like cancer which urges an improved understanding of the process. During the exosomes biogenesis, invagination of the plasma membranes forms early endosomes which mature into late endosomes and multivesicular bodies. Annexin A2 (AnxA2), a calcium dependent phospholipid binding protein, is one of the cargo proteins which gets uploading into the exosomes and impart aggressive phenotype in triple negative breast cancer (TNBC). The mechanism how AnxA2 uploads the exosomal cargo into the exosomes and releases exosomes in the tumor microenvironment remains to be unidentified. In this study, we have explored the potential mechanism for exosome biogenesis and release to target it in TNBC, which lacks the targeted based therapies. Methods: Plasmids expressing constitutive phosphomimetic (AnxA2-Y23E) and non-phosphomimetic AnxA2 (AnxA2-Y23F) mutant gene were transfected in MDA-MB-231 cells. Exosomes isolated from AnxA2-Y23E and AnxA2-Y23F mutant cells were analyzed for expression of the exosomal cargo proteins and RNAs by Western blot and RT-PCR. The number of exosomes released were analyzed by Nanotrack analysis (NTA). Mutant cells treated with Rapamycin, mTORC1(Mammalian Target of Rapamycin Complex 1) inhibitor, were analyzed for the cargo and exosomal secretion. Mutant cells were injected in nude mice to generate tumors. Serum exosomes were isolated and analyzed for cargo and number of exosome release by NTA. Results: In this study, we found that phosphorylated Annexin A2 at tyrosine 23 increases exosome secretion. It loads proteins like AnxA2, CD9 (Cluster of Differentiation 9), LC3B, and Tsg101(Tumor susceptibility gene 101), and AnxA2 and mTOR mRNA into the exosomes. Moreover, secretion and loading of cargo into the exosomes is regulated by increased phosphorylation of AnxA2 and reduced downstream mTORC1 activity. Conclusions: Phosphorylation of AnxA2 at tyrosine 23 regulates exosome secretion and cargo loading into the exosomes in TNBC.Item Role of Anti-Cancer Peptides as Immunomodulatory Agents: Potential and Design Strategy(MDPI, 2022-12-24) Tripathi, Amit K.; Vishwanatha, Jamboor K.The usage of peptide-based drugs to combat cancer is gaining significance in the pharmaceutical industry. The collateral damage caused to normal cells due to the use of chemotherapy, radiotherapy, etc. has given an impetus to the search for alternative methods of cancer treatment. For a long time, antimicrobial peptides (AMPs) have been shown to display anticancer activity. However, the immunomodulatory activity of anti-cancer peptides has not been researched very extensively. The interconnection of cancer and immune responses is well-known. Hence, a search and design of molecules that can show anti-cancer and immunomodulatory activity can be lead molecules in this field. A large number of anti-cancer peptides show good immunomodulatory activity by inhibiting the pro-inflammatory responses that assist cancer progression. Here, we thoroughly review both the naturally occurring and synthetic anti-cancer peptides that are reported to possess both anti-cancer and immunomodulatory activity. We also assess the structural and biophysical parameters that can be utilized to improve the activity. Both activities are mostly reported by different groups, however, we discuss them together to highlight their interconnection, which can be used in the future to design peptide drugs in the field of cancer therapeutics.Item Short Peptides based on the conserved regions of MIEN1 protein exhibit anti-cancer activity by targeting the MIEN1 Signaling Pathway(Elsevier B.V., 2024-01-26) Tripathi, Amit K.; Desai, Priyanka P.; Tyagi, Antariksh; Lampe, Jana B.; Srivastava, Yogesh; Donkor, Michael; Jones, Harlan P.; Dzyuba, Sergei V.; Crossley, Eric; Williams, Noelle S.; Vishwanatha, Jamboor K.Migration and invasion enhancer 1 (MIEN1) overexpression characterizes several cancers and facilitates cancer cell migration and invasion. Leveraging conserved ITAM and prenylation motifs within MIEN1, we identified potent anti-cancer peptides. Among them, bioactive peptides LA3IK and RP-7 induced pronounced transcriptomic and protein expression changes at sub-IC50 concentrations. The peptides effectively inhibited genes and proteins driving cancer cell migration, invasion, and EMT pathways, concurrently suppressing EGF-induced NF-kappaB nuclear translocation in metastatic breast cancer cells. Specifically, peptides targeted the same signal transduction pathway initiated by MIEN1. Molecular docking and circular dichroism spectroscopy indicated the formation of MIEN1-peptide complexes. The third-positioned isoleucine in LA3IK and CVIL motif in RP-7 were crucial for inhibiting breast cancer cell migration. This is evident from the limited migration inhibition observed when MDA-MB-231 cells were treated with scrambled peptides LA3IK SCR and RP-7 SCR. Additionally, LA3IK and RP-7 effectively suppressed tumor growth in an orthotopic breast cancer model. Notably, mice tolerated high peptide doses of up to 90 mg/Kg well, surpassing significantly lower doses of 5 mg/Kg intravenously (iv) and 30 mg/Kg intraperitoneally (ip) used in both in vivo pharmacokinetic studies and orthotopic mouse model assays. D-isomers of LA3IK and RP-7 showed enhanced anti-cancer activity compared to their L-isomers. D-LA3IK remained stable in mouse plasma for 24 h with 75% remaining, exhibiting superior pharmacokinetic properties over D/L-RP-7. In summary, our findings mark the first report of short peptides based on MIEN1 protein sequence capable of inhibiting cancer signaling pathways, effectively impeding cancer progression both in vitro and in vivo.