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Knockdown of RRM1 with Adenoviral Shrna Vectors to Inhibit Tumor Cell Viability and Increase Chemotherapeutic Sensitivity to Gemcitabine in Bladder Cancer Cells

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Knockdown of RRM1 with Adenoviral Shrna Vectors to Inhibit Tumor Cell Viability and Increase Chemotherapeutic Sensitivity to Gemcitabine in Bladder Cancer Cells International Journal of Molecular Sciences Article Knockdown of RRM1 with Adenoviral shRNA Vectors to Inhibit Tumor Cell Viability and Increase Chemotherapeutic Sensitivity to Gemcitabine in Bladder Cancer Cells Xia Zhang 1, Rikiya Taoka 1,*, Dage Liu 2, Yuki Matsuoka 1, Yoichiro Tohi 1 , Yoshiyuki Kakehi 1 and Mikio Sugimoto 1 1 Department of Urology, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan; [email protected] (X.Z.); [email protected] (Y.M.); [email protected] (Y.T.); [email protected] (Y.K.); [email protected] (M.S.) 2 Department of General Thoracic Surgery, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-87-891-2202 Abstract: RRM1—an important DNA replication/repair enzyme—is the primary molecular gem- citabine (GEM) target. High RRM1-expression associates with gemcitabine-resistance in various cancers and RRM1 inhibition may provide novel cancer treatment approaches. Our study eluci- dates how RRM1 inhibition affects cancer cell proliferation and influences gemcitabine-resistant bladder cancer cells. Of nine bladder cancer cell lines investigated, two RRM1 highly expressed cells, 253J and RT112, were selected for further experimentation. An RRM1-targeting shRNA was Citation: Zhang, X.; Taoka, R.; Liu, cloned into adenoviral vector, Ad-shRRM1. Gene and protein expression were investigated using D.; Matsuoka, Y.; Tohi, Y.; Kakehi, Y.; real-time PCR and western blotting. Cell proliferation rate and chemotherapeutic sensitivity to Sugimoto, M. Knockdown of RRM1 GEM were assessed by MTT assay. A human tumor xenograft model was prepared by implanting with Adenoviral shRNA Vectors to RRM1 highly expressed tumors, derived from RT112 cells, in nude mice. Infection with Ad-shRRM1 Inhibit Tumor Cell Viability and effectively downregulated RRM1 expression, significantly inhibiting cell growth in both RRM1 highly Increase Chemotherapeutic expressed tumor cells. In vivo, Ad-shRRM1 treatment had pronounced antitumor effects against Sensitivity to Gemcitabine in Bladder RRM1 highly expressed tumor xenografts (p < 0.05). Moreover, combination of Ad-shRRM1 and Cancer Cells. Int. J. Mol. Sci. 2021, 22, GEM inhibited cell proliferation in both cell lines significantly more than either treatment individ- 4102. https://doi.org/10.3390/ijms 22084102 ually. Cancer gene therapy using anti-RRM1 shRNA has pronounced antitumor effects against RRM1 highly expressed tumors, and RRM1 inhibition specifically increases bladder cancer cell GEM- Academic Editor: Georg C. Hutterer sensitivity. Ad-shRRM1/GEM combination therapy may offer new treatment options for patients with GEM-resistant bladder tumors. Received: 26 February 2021 Accepted: 12 April 2021 Keywords: RRM1; shRNA; adenoviral vector; gemcitabine; gene therapy; chemotherapeutic sensitivity Published: 15 April 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional affil- Bladder cancer is the most common malignancy of the urinary tract and the 10th iations. leading cause of malignancy worldwide [1]. Up to 75% of bladder cancers appear as frequently recurring non-muscular invasive tumors, and 15–30% of them progress to muscle-invasive cancers [2]. Radical cystectomy (RC) is the mainstay therapy for patients with muscle-invasive cancers [3]. Despite well-performed surgeries, cure rates with surgery Copyright: © 2021 by the authors. alone are only between 52.0% and 72.9%, for organ-confined disease [4,5]. With progression Licensee MDPI, Basel, Switzerland. to metastatic bladder cancer, systemic chemotherapy is considered the standard treatment. This article is an open access article Recently, gemcitabine (GEM)—an effective cytotoxic drug—was approved for the treatment distributed under the terms and of various solid tumors [6]. In addition, combination chemotherapy with GEM and cisplatin conditions of the Creative Commons (CDDP), exhibiting higher activity and fewer adverse effects, is a first-line treatment Attribution (CC BY) license (https:// for patients with metastatic bladder cancer [7]. However, while the initial response to creativecommons.org/licenses/by/ combination chemotherapy is high, long-term progression-free and overall survival rates 4.0/). Int. J. Mol. Sci. 2021, 22, 4102. https://doi.org/10.3390/ijms22084102 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 11 line treatment for patients with metastatic bladder cancer [7]. However, while the initial Int. J. Mol. Sci. 2021, 22, 4102 response to combination chemotherapy is high, long-term progression-free and2 overall of 11 survival rates are still low [8]. To improve these unsatisfactory results, new treatment drugs and strategies have gained much attention in recent years. are stillRibonucleotide low [8]. To improve reductase these M1 unsatisfactory (RRM1)—the results, large newsubunit treatment of ribonucleotide drugs and strategies reductase (RNR)—playshave gained much a central attention role in the recent process years. of DNA synthesis [9]. At the same time, RRM1 is the Ribonucleotidemolecular target reductase of GEM M1 [10]. (RRM1)—the GEM is an analogue large subunit of deoxycytidine, of ribonucleotide which reductase is phos- phorylated(RNR)—plays by deoxycytidine a central role in kinase the process and furt of DNAher by synthesis a nucleoside [9]. At monophosphate the same time, RRM1 kinase, generatingis the molecular difluorodeoxycytidine target of GEM [10 5′].-diphosphate. GEM is an analogueThis phosphorylated of deoxycytidine, product which binds is to thephosphorylated binding site and by inactivates deoxycytidine the kinaseRRM1 andsubunit, further whereas by anucleoside the triphosphorylated monophosphate form is kinase, generating difluorodeoxycytidine 50-diphosphate. This phosphorylated product incorporated into DNA chain and inhibits DNA synthesis [11]. Studies have shown that binds to the binding site and inactivates the RRM1 subunit, whereas the triphosphorylated GEM-resistance in various cancers is associated with an increased expression of RRM1 form is incorporated into DNA chain and inhibits DNA synthesis [11]. Studies have [12,13]. Several clinical studies have confirmed the relationship between high expression shown that GEM-resistance in various cancers is associated with an increased expression ofof RRM1 RRM1 and [12 ,adverse13]. Several clinical clinical outcomes studies in have pati confirmedents with advanced the relationship bladder between cancer high[14,15]. Ofexpression such patients of RRM1 who andwere adverse treated clinicalwith first-line outcomes GEM in patientsplus platinum with advanced combination bladder chem- otherapy,cancer [14 those,15]. with Of such low patients expression who of were RRM1 treated showed with a first-linehigher treatment GEM plus response platinum rate [16],combination whilst those chemotherapy, with high those RRM1 with expression low expression had a of significantly RRM1 showed greater a higher risk treatment of disease progressionresponse rate and [16 ],death whilst [15]. those Th witherefore, high RRM1inhibiting expression RRM1 hadmay a significantlybecome a new greater treatment risk strategyof disease in patients progression with and bladder death cancer, [15]. Therefore, not only to inhibiting inhibit cell RRM1 viability may but become also to a newreduce GEM-resistance.treatment strategy in patients with bladder cancer, not only to inhibit cell viability but also to reducePreviously, GEM-resistance. we demonstrated that an adenoviral vector expressing short hairpin RNA thatPreviously, targets weRRM1 demonstrated (Ad-shRRM1) that anhad adenoviral antiproliferative vector expressing activity against short hairpin non-small RNA cell lungthat targetscancer RRM1cells [17]. (Ad-shRRM1) In the present had antiproliferative study, we further activity explored against non-smallthe potential cell lungof Ad- shRRM1,cancer cells this [17 time]. In in the bladder present study,cancer we cells further and exploredfound that the Ad-shRRM1 potential of Ad-shRRM1, effectively thisdown- time in bladder cancer cells and found that Ad-shRRM1 effectively downregulated RRM1 regulated RRM1 expression, resulting in suppression of bladder cancer cell proliferation expression, resulting in suppression of bladder cancer cell proliferation in vitro and in vivo. in vitro and in vivo. Moreover, combined treatment with Ad-shRRM1 and GEM showed Moreover, combined treatment with Ad-shRRM1 and GEM showed effective antitumor effective antitumor activity in bladder cancer cells. activity in bladder cancer cells. 2.2. Results Results 2.1.2.1. RRM1 RRM1 Gene Gene Expression Expression Isis Higher inin BladderBladder Cancer Cancer Cell Cell Lines Lines TheThe normalized normalized RRM1 gene expressionexpression ratio ratio was was evaluated evaluated in in nine nine human human bladder bladder cancer,cancer, six six lung lung cancer, cancer, andand sixsix malignantmalignant mesothelioma mesothelioma cells cells (Figure (Figure1A). 1A). RRM1 RRM1 gene gene expressionexpression varied varied from from 28.6% (T24)(T24) toto 153.5%153.5% (J82) (J82) (median: (median: 116.9%) 116.9%) in in bladder bladder cancer cancer cells.cells. Compared Compared to to the the lung cancercancer oror malignantmalignant mesothelioma mesothelioma cell cell lines, lines, bladder
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