Browsing by Author "Crooks, Peter A."

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    Aging-associated skeletal muscle defects in HER2/Neu transgenic mammary tumor model
    (Wiley, 2021) Wang, Ruizhong; Kumar, Brijesh; Bhat-Nakshatri, Poornima; Prasad, Mayuri S.; Jacobsen, Max H.; Ovalle, Gabriela; Maguire, Calli; Sandusky, George; Trivedi, Trupti; Mohammad, Khalid S.; Guise, Theresa; Penthala, Narsimha R.; Crooks, Peter A.; Liu, Jianguo; Zimmers, Teresa; Nakshatri, Harikrishna; Surgery, School of Medicine
    Background: Loss of skeletal muscle volume and resulting in functional limitations are poor prognostic markers in breast cancer patients. Several molecular defects in skeletal muscle including reduced MyoD levels and increased protein turn over due to enhanced proteosomal activity have been suggested as causes of skeletal muscle loss in cancer patients. However, it is unknown whether molecular defects in skeletal muscle are dependent on tumor etiology. Methods: We characterized functional and molecular defects of skeletal muscle in MMTV-Neu (Neu+) mice (n= 6-12), an animal model that represents HER2+ human breast cancer, and compared the results with well-characterized luminal B breast cancer model MMTV-PyMT (PyMT+). Functional studies such as grip strength, rotarod performance, and ex vivo muscle contraction were performed to measure the effects of cancer on skeletal muscle. Expression of muscle-enriched genes and microRNAs as well as circulating cytokines/chemokines were measured. Since NF-κB pathway plays a significant role in skeletal muscle defects, the ability of NF-κB inhibitor dimethylaminoparthenolide (DMAPT) to reverse skeletal muscle defects was examined. Results: Neu+ mice showed skeletal muscle defects similar to accelerated aging. Compared to age and sex-matched wild type mice, Neu+ tumor-bearing mice had lower grip strength (202±6.9 vs. 179±6.8 g grip force, p=0.0069) and impaired rotarod performance (108±12.1 vs. 30±3.9 seconds, P<0.0001), which was consistent with reduced muscle contractibility (p<0.0001). Skeletal muscle of Neu+ mice (n=6) contained lower levels of CD82+ (16.2±2.9 vs 9.0±1.6) and CD54+ (3.8±0.5 vs 2.4±0.4) muscle stem and progenitor cells (p<0.05), suggesting impaired capacity of muscle regeneration, which was accompanied by decreased MyoD, p53 and miR-486 expression in muscles (p<0.05). Unlike PyMT+ mice, which showed skeletal muscle mitochondrial defects including reduced mitochondria levels and Pgc1β, Neu+ mice displayed accelerated aging-associated changes including muscle fiber shrinkage and increased extracellular matrix deposition. Circulating "aging factor" and cachexia and fibromyalgia-associated chemokine Ccl11 was elevated in Neu+ mice (1439.56±514 vs. 1950±345 pg/ml, p<0.05). Treatment of Neu+ mice with DMAPT significantly restored grip strength (205±6 g force), rotarod performance (74±8.5 seconds), reversed molecular alterations associated with skeletal muscle aging, reduced circulating Ccl11 (1083.26 ±478 pg/ml), and improved animal survival. Conclusions: These results suggest that breast cancer subtype has a specific impact on the type of molecular and structure changes in skeletal muscle, which needs to be taken into consideration while designing therapies to reduce breast cancer-induced skeletal muscle loss and functional limitations.
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    Antitumor Properties of Novel Sesquiterpene Lactone Analogs as NFκB Inhibitors that Bind to the IKKβ Ubiquitin-like Domain (ULD)
    (Elsevier, 2021) Penthala, Narsimha R.; Balasubramaniam, Meenakshisundaram; Dachavaram, Soma Shekar; Morris, Earl J.; Bhat-Nakshatri, Poornima; Ponder, Jessica; Jordan, Craig T.; Nakshatri, Harikrishna; Crooks, Peter A.; Surgery, School of Medicine
    Melampomagnolide B (MMB, 3) is a parthenolide (PTL, 1) based sesquiterpene lactone that has been used as a template for the synthesis of a plethora of lead anticancer agents owing to its reactive C-10 primary hydroxyl group. Such compounds have been shown to inhibit the IKKβ subunit, preventing phosphorylation of the cytoplasmic IκB inhibitory complex. The present study focuses on the synthesis and in vitro antitumor properties of novel benzyl and phenethyl carbamates of MMB (7a-7k). Screening of these MMB carbamates identified analogs with potent growth inhibition properties against a panel of 60 human cancer cell lines (71% of the molecules screened had GI50 values <2 μM). Two analogs, the benzyl carbamate 7b and the phenethyl carbamate7k, were the most active compounds. Lead compound 7b inhibited cell proliferation in M9 ENL AML cells, and in TMD-231, OV-MD-231 and SUM149 breast cancer cell lines. Interestingly, mechanistic studies showed that 7b did not inhibit p65 phosphorylation in M9 ENL AML and OV-MD-231 cells, but did inhibit phophorylation of both p65 and IκBα in SUM149 cells. 7b also reduced NFκB binding to DNA in both OV-MD-231 and SUM149 cells. Molecular docking studies indicated that 7b and 7k are both predicted to interact with the ubiquitin-like domain (ULD) of the IKKβ subunit. These data suggest that in SUM149 cells, 7b is likely acting as an allosteric inhibitor of IKKβ, whereas in M9 ENL AML and OV-MD-231 cells 7b is able to inhibit an event after IκB/p65/p50 phosphorylation by IKKβ that leads to inhibition of NFκB activation and reduction in NFκB-DNA binding. Analog 7b was by far the most potent compound in either carbamate series, and was considered an important lead compound for further optimization and development as an anticancer agent.
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    DMAPT inhibits NF-κB activity and increases sensitivity of prostate cancer cells to X-rays in vitro and in tumor xenografts in vivo
    (Elsevier, 2017-11) Mendonca, Marc S.; Turchan, William T.; Alpuche, Melanie E.; Watson, Christopher N.; Estabrook, Neil C.; Chin-Sinex, Helen; Shapiro, Jeremy B.; Imasuen-Williams, Imade E.; Rangel, Gabriel; Gilley, David P.; Huda, Nazmul; Crooks, Peter A.; Shapiro, Ronald H.; Department of Radiation Oncology, School of Medicine
    Constitutive activation of the pro-survival transcription factor NF-κB has been associated with resistance to both chemotherapy and radiation therapy in many human cancers, including prostate cancer. Our lab and others have demonstrated that the natural product parthenolide can inhibit NF-κB activity and sensitize PC-3 prostate cancers cells to X-rays in vitro; however, parthenolide has poor bioavailability in vivo and therefore has little clinical utility in this regard. We show here that treatment of PC-3 and DU145 human prostate cancer cells with dimethylaminoparthenolide (DMAPT), a parthenolide derivative with increased bioavailability, inhibits constitutive and radiation-induced NF-κB binding activity and slows prostate cancer cell growth. We also show that DMAPT increases single and fractionated X-ray-induced killing of prostate cancer cells through inhibition of DNA double strand break repair and also that DMAPT-induced radiosensitization is, at least partially, dependent upon the alteration of intracellular thiol reduction-oxidation chemistry. Finally, we demonstrate that the treatment of PC-3 prostate tumor xenografts with oral DMAPT in addition to radiation therapy significantly decreases tumor growth and results in significantly smaller tumor volumes compared to xenografts treated with either DMAPT or radiation therapy alone, suggesting that DMAPT might have a potential clinical role as a radiosensitizing agent in the treatment of prostate cancer.
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    An improved model of ethanol and nicotine co-use in female P rats: Effects of naltrexone, varenicline, and the selective nicotinic α6β2* antagonist r-bPiDI
    (Elsevier, 2018-12) Maggio, Sarah E.; Saunders, Meredith A.; Nixon, Kimberly; Prendergast, Mark A.; Zheng, Guangrong; Crooks, Peter A.; Dwoskin, Linda P.; Bell, Richard L.; Bardo, Michael T.; Psychiatry, School of Medicine
    Background Although pharmacotherapies are available for alcohol (EtOH) or tobacco use disorders individually, it may be possible to develop a single pharmacotherapy to treat heavy drinking tobacco smokers by capitalizing on the commonalities in their mechanisms of action. Methods Female alcohol-preferring (P) rats were trained for EtOH drinking and nicotine self-administration in two phases: (1) EtOH alone (0 vs. 15% EtOH, 2-bottle choice) and (2) concomitant access, during which EtOH access continued with access to nicotine (0.03 mg/kg/infusion, i.v.) using a 2-lever choice procedure (active vs. inactive lever) in which the fixed ratio (FR) requirement was gradually increased to FR30. When stable co-use was obtained, rats were pretreated with varying doses of naltrexone, varenicline, or r-bPiDI, an α6β2* subtype-selective nicotinic acetylcholine receptor antagonist shown previously to reduce nicotine self-administration. Results While EtOH intake was initially suppressed in phase 2 (co-use), pharmacologically relevant intake for both substances was achieved by raising the “price” of nicotine to FR30. In phase 2, naltrexone decreased EtOH and water consumption but not nicotine intake; in contrast, naltrexone in phase 1 (EtOH only) did not significantly alter EtOH intake. Varenicline and r-bPiDI in phase 2 both decreased nicotine self-administration and inactive lever pressing, but neither altered EtOH or water consumption. Conclusions These results indicate that increasing the “price” of nicotine increases EtOH intake during co-use. Additionally, the efficacy of naltrexone, varenicline, and r-bPiDI was specific to either EtOH or nicotine, with no efficacy for co-use. Nevertheless, future studies on combining these treatments may reveal synergistic efficacy.
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    MMB triazole analogs are potent NF-κB inhibitors and anti-cancer agents against both hematological and solid tumor cells
    (Elsevier, 2018-09) Janganati, Venumadhav; Ponder, Jessica; Balasubramaniam, Meenakshisundaram; Bhat-Nakshatri, Poornima; Bar, Eli E.; Nakshatri, Harikrishna; Jordan, Craig T.; Crooks, Peter A.; Surgery, School of Medicine
    Triazole derivatives of melampomagnolide B (MMB) have been synthesized via click chemistry methodologies and screened against a panel of 60 human cancer cell lines. Several derivatives showed promising anti-cancer activity, affording growth inhibition (GI50) values in the nanomolar range (GI50 = 0.02–0.99 μM). Lead compound 7h exhibited EC50 values of 400 nM and 700 nM, respectively, against two AML clinical specimens. Compound 7h was significantly more potent than parthenolide as an inhibitor of p65 phosphorylation in both hematological and solid tumor cell lines, indicating its ability to inhibit the NF-κB pathway. In TMD-231 breast cancer cells, treatment with 7h reduced DNA binding activity of NF-κB through inhibition of IKK-β mediated p65 phosphorylation and caused elevation of basal IκBα levels through inhibition of constitutive IκBα turnover and NF-κB activation. Molecular docking and dynamic modeling studies indicated that 7h interacts with the kinase domain of the monomeric IKKβ subunit, leading to inhibition of IKKβ activation, and compromising phosphorylation of downstream targets of the NF-κB pathway; dynamic modeling studies show that this interaction also causes unwinding of the α-helix of the NEMO binding site on IKKβ. Molecular docking studies with 10, a water-soluble analog of 7h, demonstrate that this analog interacts with the dimerization/oligomerization domain of monomeric IKKβ and may inhibit oligomer formation and subsequent autophosphorylation. Sesquiterpene lactones 7h and 10 are considered ideal candidates for potential clinical development.
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    Pharmacological Dual Inhibition of Tumor and Tumor-Induced Functional Limitations in a Transgenic Model of Breast Cancer
    (American Association for Cancer Research, 2017-12) Wang, Ruizhong; Bhat-Nakshatri, Poornima; Padua, Maria B.; Prasad, Mayuri S.; Anjanappa, Manjushree; Jacobson, Max; Finnearty, Courtney; Sefcsik, Victoria; McElyea, Kyle; Redmond, Rachael; Sandusky, George; Penthala, Narsimha; Crooks, Peter A.; Liu, Jianguo; Zimmers, Teresa A.; Nakshatri, Harikrishna; Surgery, School of Medicine
    Breast cancer progression is associated with systemic effects, including functional limitations and sarcopenia without the appearance of overt cachexia. Autocrine/paracrine actions of cytokines/chemokines produced by cancer cells mediate cancer progression and functional limitations. The cytokine-inducible transcription factor NF-κB could be central to this process, as it displays oncogenic functions and is integral to the Pax7:MyoD:Pgc-1β:miR-486 myogenesis axis. We tested this possibility using the MMTV-PyMT transgenic mammary tumor model and the NF-κB inhibitor dimethylaminoparthenolide (DMAPT). We observed deteriorating physical and functional conditions in PyMT+ mice with disease progression. Compared with wild-type mice, tumor-bearing PyMT+ mice showed decreased fat mass, impaired rotarod performance, and reduced grip strength as well as increased extracellular matrix (ECM) deposition in muscle. Contrary to acute cachexia models described in the literature, mammary tumor progression was associated with reduction in skeletal muscle stem/satellite-specific transcription factor Pax7. Additionally, we observed tumor-induced reduction in Pgc-1β in muscle, which controls mitochondrial biogenesis. DMAPT treatment starting at 6 to 8 weeks age prior to mammary tumor occurrence delayed mammary tumor onset and tumor growth rates without affecting metastasis. DMAPT overcame cancer-induced functional limitations and improved survival, which was accompanied with restoration of Pax7, Pgc-1β, and mitochondria levels and reduced ECM levels in skeletal muscles. In addition, DMAPT restored circulating levels of 6 out of 13 cancer-associated cytokines/chemokines changes to levels seen in healthy animals. These results reveal a pharmacological approach for overcoming cancer-induced functional limitations, and the above-noted cancer/drug-induced changes in muscle gene expression could be utilized as biomarkers of functional limitations.