Human fibrosarcoma cells selected for very-high doxorubicin resistance, acquire trabectedin and eribulin cross-resistance, remain sensitive to recombinant methioninase, and have increased c-MYC expression

Human fibrosarcoma cells selected for very-high doxorubicin resistance, acquire trabectedin and eribulin cross-resistance, remain sensitive to recombinant methioninase, and have increased c-MYC expression | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Human fibrosarcoma cells selected for very-high doxorubicin resistance, acquire trabectedin and eribulin cross-resistance, remain sensitive to recombinant methioninase, and have increased c-MYC expression View ORCID Profile Sei Morinaga , Qinghong Han , Kohei Mizuta , Byung Mo Kang , Michael Bouvet , Norio Yamamoto , Katsuhiro Hayashi , Hiroaki Kimura , Shinji Miwa , Kentaro Igarashi , Takashi Higuchi , Hiroyuki Tsuchiya , Satoru Demura , Robert M. Hoffman doi: https://doi.org/10.1101/2025.06.07.658455 Sei Morinaga 1 AntiCancer Inc. , San Diego, CA, U.S.A 2 Department of Surgery, University of California , San Diego, CA, U.S.A 3 Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kanazawa University , Kanazawa, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sei Morinaga For correspondence: reddchicke{at}yahoo.co.jp Qinghong Han 1 AntiCancer Inc. , San Diego, CA, U.S.A Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kohei Mizuta 1 AntiCancer Inc. , San Diego, CA, U.S.A 2 Department of Surgery, University of California , San Diego, CA, U.S.A Find this author on Google Scholar Find this author on PubMed Search for this author on this site Byung Mo Kang 1 AntiCancer Inc. , San Diego, CA, U.S.A 2 Department of Surgery, University of California , San Diego, CA, U.S.A Find this author on Google Scholar Find this author on PubMed Search for this author on this site Michael Bouvet 2 Department of Surgery, University of California , San Diego, CA, U.S.A Find this author on Google Scholar Find this author on PubMed Search for this author on this site Norio Yamamoto 3 Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kanazawa University , Kanazawa, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Katsuhiro Hayashi 3 Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kanazawa University , Kanazawa, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hiroaki Kimura 3 Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kanazawa University , Kanazawa, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Shinji Miwa 3 Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kanazawa University , Kanazawa, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kentaro Igarashi 3 Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kanazawa University , Kanazawa, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Takashi Higuchi 3 Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kanazawa University , Kanazawa, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hiroyuki Tsuchiya 3 Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kanazawa University , Kanazawa, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Satoru Demura 3 Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kanazawa University , Kanazawa, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Robert M. Hoffman 1 AntiCancer Inc. , San Diego, CA, U.S.A 2 Department of Surgery, University of California , San Diego, CA, U.S.A Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abstract Full Text Info/History Metrics Preview PDF Abstract Doxorubicin is first-line chemotherapy for soft tissue sarcoma; however, the development of drug resistance limits its efficacy. The purpose of the present study was to select very-high doxorubicin-resistant (VHDR) HT1080 fibrosarcoma cells, determine cross-resistance to second-line chemotherapy drugs, determine maintenance of sensitivity to recombinant methioninase (r-METase) alone and in combination with doxorubicin, and measure the level of c-MYC expression. VHDR-HT1080 cells were generated by cultivating HT-1080 cells in a series of step-wise progressively higher concentrations of doxorubicin, ranging from 8 nM to 15 µM, an 1875-fold increase, over a five-month period. The WST-8 reagent was used to assess cell viability. Four groups of in vitro drug-sensitivity tests were conducted, which involved both parental HT1080 and VHDR-HT1080 cells: 1) doxorubicin alone; 2) rMETase alone; 3) a combination of doxorubicin and rMETase; and 4) untreated control. The cross-resistance of VHDR-HT1080 cells to eribulin, trabectedin, gemcitabine and docetaxel was determined. The c-MYC levels in HT1080 and VHDR-HT1080 cells were measured using Western blotting. Doxorubicin had an IC 50 of 3.3 µM against HT1080 cells and 38.2 µM against VHDR-HT1080 cells an 11.6-fold increase. The rMETase IC 50 value for HT1080 was 0.75 U/ml and 0.59 U/ml for VHDR-HT1080. rMETase sensitized VHDR-HT1080 cells to doxorubicin. VHDR-HT1080 cells were cross-resistant to trabectedin 8.9-fold and cross-resistant to eribulin 1.87-fold compared to parental HT1080 cells. c-MYC expression was 8.4 times higher in VHDR-HT1080 cells compared to HT-1080 cells. The present results suggest rMETase may be used as a future clinical strategy to overcome super-doxorubicin resistance in soft tissue sarcoma. Introduction Soft tissue sarcomas (STS) are a heterogeneous group of malignant tumors that arise from mesenchymal tissues and account for approximately 1% of all adult cancers ( 1 ). Doxorubicin is a first-line chemotherapy for STS ( 2 ). The development of acquired drug resistance to doxorubicin has limited the clinical efficacy of this drug. High levels of c-MYC expression correlate with poor prognosis and increased resistance to chemotherapeutic agents in various cancer types, including STS ( 3 , 4 ). Metastatic STS remains a recalcitrant disease in need of improved therapy. Methionine addiction is a general and fundamental hallmark of cancer termed the Hoffman effect ( 5 , 6 ). Methionine restriction, including recombinant methioninase (rMETase), selectively arrests cancer cells in the late-S/G 2 phase of the cell cycle ( 7 , 8 ) and has been shown to increase the efficacy of all types of chemotherapy drugs which target cells in late-S/G 2 ( 9 – 16 ). Recently we have studied the rMETase sensitivity of drug-resistant sarcoma cells ( 16 – 22 ). In the present study, we aimed to establish a very high doxorubicin-resistant HT1080 fibrosarcoma cell line (VHDR-HT1080) and investigate cross-resistance to other drugs, the maintenance of rMETase sensitivity and the level of c-MYC expression. Materials and Methods Cell culture The American Type Culture Collection (Manassas, VA, USA) the provided HT1080 cell line. Cells were grown in DMEM with 10% FBS and 1 IU/ml penicillin and streptomycin. Reagents Bedford Laboratories (Bedford, OH, USA) provided doxorubicin. AntiCancer Inc. (San Diego, CA, USA) produced rMETase. The process of producing rMETase has already been published ( 8 ): Briefly, the Pseudomonas putida methioninase gene was cloned in E. coli . which was fermented to produce recombinant methioninase which was purified with a new step, diethylaminoethyl-sepharose fast-flow ion-exchange column chromatography. Establishment of very-high doxorubicin-resistant HT1080 (VHDR-HT1080) cells Over the course of five months, HT1080 cells were cultured in doxorubicin concentrations that increased stepwise from 8 nM to 15 µM, an 1875-fold increase. Drug sensitivity assay 1: IC 50 Cell viability was assessed using the WST-8 reagent (Dojindo Laboratory, Kumamoto, Japan). HT1080 or VHDR-HT1080 cells were cultured in 96-well plates at a concentration of 3×10 3 cells per well in DMEM (100 μl/well). After that, the plates were incubated overnight at 37°C. The cells were treated for 72 hours with either rMETase at concentrations ranging from 0.5 U/ml to 8 U/ml or doxorubicin at concentrations ranging from 1 µM to 40 µM. Each well received 10 μl of the WST-8 solution following the culture period. The plates were then incubated at 37°C for an additional hour. In a microplate reader (SUNRISE: TECAN, Mannedorf, Switzerland), the absorption of cells treated with WST-8 was measured at 450 nM. Microsoft Excel for Mac 2016 version 15.52 (Microsoft, Redmond, Washington, United States) was used to create the drug sensitivity curves. ImageJ version 1.53k (National Institutes of Health, Bethesda, MD, USA) was used to calculate the half-maximal inhibitory concentration (IC 50 ) values. Each experiment was carried out twice, in triplicate. Combination drug sensitivity assay 96-well plates were seeded with 3×10 3 VHDR-HT1080 cells. The cells received the following treatment after 24 hours: 1) No treatment; 2) Doxorubicin alone; 3) rMETase alone; and 4) the combination of rMETase and doxorubicin. After 72 hours, cell viability was determined using the WST-8 regent in triplicate. Cross-resistance assay The same protocol as in drug sensitivity assay 1 was used. Second-line drugs for soft tissue sarcoma, eribulin (Eisai Inc., Nutley, NJ, USA) (0.5-8 nM), trabectedin (PharmaMar, Horsham, PA, USA) (1-40 nM), gemcitabine (BluePoint Laboratories, Little Island, Cork, Munster, Ireland) (4-64 nM), and docetaxel (Accord Healthcare Inc., Durham, NC, USA) (1-16 nM), were used to determine cross-resistance of VHDR-HT1080 cells. The IC 50 values of each drug to HT1080 and VHDR-HT1080 were determined. Cross-resistance to a drug was defined as being present when there was a increase of 1.5-fold or more in the IC 50 . Western immunoblotting Proteins were extracted from HT1080 and VHDR-HT1080 cells using RIPA Lysis Buffer and Extraction Buffer (Thermo Fisher Scientific, Waltham, MA, USA) and 1% Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific). 10% SDS-PAGE gels were loaded with protein samples. The samples were then transferred to polyvinylidene difluoride (PVDF) membranes with a thickness of 0.45 μm (GE Healthcare, Chicago, IL, USA). Membrane blocking was done using Bullet Blocking One for Western Blotting (Nakalai Tesque, Inc., Kyoto, Japan). The anti-c-MYC antibody was obtained from Proteintech (1:2,000, #10828-1-AP, Rosemont, IL, USA) as well as β-Actin (20536-1-AP, 1:1,000). Horseradish-peroxidase-conjugated anti-rabbit IgG (1:5,000, #SA00001-2, Proteintech) were used as secondary antibodies. The western blot was scanned using the Clarity Western ECL Substrate (Bio-Rad Laboratories, Hercules, California, USA) and UVP ChemStudio (Analytik Jena, Upland, CA, USA). The experiments were conducted three times. Statistical analyses were conducted using EZR software (Jichi Medical University, Saitama, Japan) ( 24 ). The Welch’s t-test and Tukey-Kramer analysis were employed to ascertain the correlation between the variables. Statistically significant p -values were defined as less than 0.05 ( 25 ). Results Establishment of very-high doxorubicin-resistant HT1080 cells Very-highly doxorubicin-resistant cells (VHDR-HT1080) were selected from HT1080 cells by culturing them in doxorubicin, increasing stepwise from 8 nM to 15 µM, an 1875-fold increase over a period of 5 months. The HT1080 IC 50 of doxorubicin was 3.3 µM [data from ( 16 )], compared to 38.2 µM for VHDR-HT1080 cells. VHDR-HT1080 cells were 11.6 times more resistant to doxorubicin than the parental HT1080 cells ( Figure 1 ). Download figure Open in new tab Figure 1: IC 50 of doxorubicin on VHDR-HT1080 cells. Please see Materials and Methods for details. Results are shown as mean ± standard deviation. Determination of IC 50 of rMETase alone on HT1080 and VHDR-HT1080 The HT1080 IC 50 of rMETase was 0.75 U/ml [data from ( 10 )], compared to the VHDR-HT1080 IC 50 of 0.59 U/ml ( Figure 2 ). Download figure Open in new tab Figure 2: IC 50 of rMETase on VHDR-HT1080 cells. Please see Materials and Methods for details. Results are shown as mean ± standard deviation. VHDR-HT1080 Cross-resistance of VHDR-HT1080 cells to second-line STS chemotherapy drugs The IC 50 values for HT1080 and VHDR-HT1080 cells were 0.15 nM [data from ( 10 )]and 0.28 nM for eribulin, respectively; 3.3 nM [data from ( 15 )] and 29.3 nM for trabectedin, respectively; 12.8 nM [data from ( 25 )] and 13.6 nM for gemcitabine, respectively; and 1.68 nM [data from ( 19 )] and 1.83 nM for docetaxel, respectively ( Table I ). View this table: View inline View popup Download powerpoint Table 1. IC 50 of HT1080 and very-highly doxorubicin-resistant HT1080 (VHDR-HT1080) cells to eribulin, trabectedin, gemcitabine or docetaxel. VHDR-HT1080 cells had cross-resistance to trabectedin and eribulin. Please see Materials and Methods for details. Efficacy of rMETase combined with doxorubicin on VHDR-HT1080 cells The IC 50 of rMETase for VHDR-HT1080 (0.59 U/ml) combined with the IC 50 of doxorubicin for HT1080 (3.3 µM) inhibited VHDR cells 73.4% compared to determined control, 69.7% compared to doxorubicin alone and 15.8% compared to rMETase alone ( p <0.05) ( Figure 3 ). Download figure Open in new tab Figure 3: rMETase sensitized very high doxorubicin-resistant HT1080 (VHDR-HT1080) fibrosarcoma cells to doxorubicin. Control (DMEM); doxorubicin [3.3 µM (IC 50 of HT1080)]; rMETase [0.59 U/ml (IC 50 of VHDR-HT1080)]; doxorubicin [3.3 µM (IC 50 of HT1080)] and rMETase [0.59 U/ml (IC 50 of VHDR-HT1080)]. Data are shown as mean ± standard deviation. Please see Materials and Methods for details. Western blotting of c-MYC c-MYC expression in VHDR-HT1080 cells increased 8.4-fold compared to HT1080 cells ( p <0.05) ( Figure 4 ). Download figure Open in new tab Figure 4. Expression of c-MYC in HT1080 and very-highly doxorubicin-resistant HT1080 (VHDR-HT1080) fibrosarcoma cells. A) Western blot of c-MYC expression in HT1080 and VHDR-HT1080 cells. B) Quantitation of c-MYC expression in HT1080 and VHDR-HT1080 cells. Data shown are representative of three different Western blots. Please see Materials and Methods for details. Discussion VHDR-HT1080 was established by selecting HT1080 cells in stepwise increasing concentrations of doxorubicin (1875-fold) over five months. VHDR-HT1080 cells acquired very-high resistance to doxorubicin, with an IC 50 value 11.6-fold greater than that of parental HT1080 cells. VHDR-HT1080 cells were cross-resistant to second-line therapy, trabectedin by 8.9-fold and to eribulin by 1.87-fold, suggesting a shared resistance mechanism. The IC 50 of rMETase was similar in HT1080 and VHDR-HT1080 that indicates the acquisition of very high doxorubicin resistance did not affect rMETase sensitivity. rMETase sensitized VHDR-HT1080 cells 19.8-fold to doxorubicin. These results suggest that rMETase may be effective clinically to overcome doxorubicin resistance in STS. Further studies are needed to determine if c-MYC overexpression is linked to very high doxorubicin resistance in STS as well as the mechanism of cross-resistance to second-line STS chemotherapy in VHDR cells. The present results suggest that combining rMETase and doxorubicin may be a clinical strategy to overcome clinical doxorubicin-resistant STS that are also cross-resistant to second-line STS drugs. Conflicts of Interest The Authors declare no competing interests in relation to this study. Authors’ Contributions Conceptualization: SM KM RMH Data curation: SM RMH Formal analysis: SM KM BMK MB NY KH HK SM KI TH HT SD QH Investigation: SM KM QH Methodology: SM KM RMH Project administration: RMH Validation: KM BMK Writing– original draft: SM Writing– review & editing: SM, RMH. Acknowledgements This article is dedicated to the memory of A.R. Moossa, MD, Sun Lee, MD, Richard W. Erbe, MD, Professor Philip Miles, Professor Gordon H. Sato, Professor Li Jiaxi, Masaki Kitajima, MD, Joseph R. Bertino, MD, Shigeo Yagi, PhD, J.A.R Mead, Ph.D., Eugene P. Frenkel, MD, Professor Lev Bergelson, Professor Sheldon Penman, Professor John R. Raper, Joseph Leighton, MD and John Mendelsohn, MD. References 1. ↵ Gamboa AC , Gronchi A , Cardona K. Soft-tissue sarcoma in adults: An update on the current state of histiotype-specific management in an era of personalized medicine . CA Cancer J Clin . 2020 ; 70 ( 3 ): 200 – 229 . doi: 10.3322/caac.21605 . PMID: 32275330 . 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Elevated-c-MYC-expressing fibrosarcoma cells with acquired gemcitabine resistance remain sensitive to recombinant methioninase: a potential clinical strategy for a recalcitrant disease . Cancer Diagn Progn . 2025 ; 5 ( 1 ): 8 – 14 . doi: 10.21873/cdp.10405 . PMID: 39758233 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted June 11, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Human fibrosarcoma cells selected for very-high doxorubicin resistance, acquire trabectedin and eribulin cross-resistance, remain sensitive to recombinant methioninase, and have increased c-MYC expression Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Human fibrosarcoma cells selected for very-high doxorubicin resistance, acquire trabectedin and eribulin cross-resistance, remain sensitive to recombinant methioninase, and have increased c-MYC expression Sei Morinaga , Qinghong Han , Kohei Mizuta , Byung Mo Kang , Michael Bouvet , Norio Yamamoto , Katsuhiro Hayashi , Hiroaki Kimura , Shinji Miwa , Kentaro Igarashi , Takashi Higuchi , Hiroyuki Tsuchiya , Satoru Demura , Robert M. Hoffman bioRxiv 2025.06.07.658455; doi: https://doi.org/10.1101/2025.06.07.658455 Share This Article: Copy Citation Tools Human fibrosarcoma cells selected for very-high doxorubicin resistance, acquire trabectedin and eribulin cross-resistance, remain sensitive to recombinant methioninase, and have increased c-MYC expression Sei Morinaga , Qinghong Han , Kohei Mizuta , Byung Mo Kang , Michael Bouvet , Norio Yamamoto , Katsuhiro Hayashi , Hiroaki Kimura , Shinji Miwa , Kentaro Igarashi , Takashi Higuchi , Hiroyuki Tsuchiya , Satoru Demura , Robert M. 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