{"paper_id":"72b0b799-6a38-409a-af34-7568ea60c2d0","body_text":"https://doi.org/10.1530/JOE-17-0700\nhttp://joe.endocrinology-journals.org © 2018 Society for Endocrinology\nPrinted in Great Britain\nPublished by Bioscientifica Ltd.\nJournal of \nEndocrinology\n237:3\n255–269Y J Cho, J E Lee et al. Bufalin suppresses \nendometriosis\n10.1530/JOE-17-0700\nRESEARCH\nBufalin suppresses endometriosis progression \nby inducing pyroptosis and apoptosis\nYeon Jean Cho1,2,*, Jiyeun E Lee1,*, Mi Jin Park1, Bert W O’Malley1,3 and Sang Jun Han1,3\n1Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA\n2Department of Obstetrics and Gynecology, Dong-A University, College of Medicine, Busan, Republic of Korea\n3Center for Reproductive Medicine, Baylor College of Medicine, Houston, Texas, USA\nCorrespondence should be addressed to S J Han: sjhan@bcm.edu\n*(Y J Cho and J Lee contributed equally to this work)\nAbstract\nThe steroid receptor coactivator (SRC)-1 isoform/estrogen receptor (ER)- β axis has \nan essential role in endometriosis progression. In this context, therefore, bufalin \nwas employed as a ‘tool compound’ to evaluate inhibitors of SRC in alternative \nendometriosis treatment. Bufalin effectively suppressed the growth of primary  \nhuman endometrial stroma cells isolated from endometriosis patients compared to \nwomen without endometriosis and immortalized human endometrial epithelial and \nstromal cells expressing the SRC-1 isoform compared to their parental cells in vitro . \nIn vivo , compared to the vehicle, bufalin treatment significantly suppressed the \ngrowth of endometriotic lesions in mice with surgically induced endometriosis because \nbufalin disrupted the functional axis of SRC-1 isoform/ER β by increasing SRC-1 isoform \nprotein stability, hyperactivating the transcriptional activity of the SRC-1 isoform and \ndegrading the ER β protein by proteasome 26S subunit, non-ATPase 2 in  \nendometriotic lesions. Bufalin treatment elevated the apoptosis signaling in  \nepithelial cells of endometriotic lesions. In stromal cells of endometriotic lesions, \nbufalin treatment increased the levels of pyroptosis markers (caspase 1 and the  \nactive form of interleukin 1 β) and reduced proliferation. In addition, bufalin  \ntreatment increased the expression levels of endoplasmic reticulum-stress (ERS) \nmarkers (PKR-like ER kinase, protein disulfide isomerase and binding immunoglobulin) \nin endometriotic lesions. Collectively, the bufalin-induced disruption of the SRC-1 \nisoform/ER β axis might induce apoptosis, pyroptosis and ERS signaling in endometriotic \nlesions, causing the suppression of endometriosis. Therefore, future generations \nof SRC-modulators could be employed as an alternative medical approach for \nendometriosis treatment.\nIntroduction\nAs an estrogen-dependent pro-inflammatory disease, \nendometriosis is defined as the colonization and growth \nof endometrial tissues at anatomic sites outside of the \nuterine cavity, primarily in the pelvic peritoneum and \novaries ( Bulun 2009 ). Up to 10% of reproductive-aged \nwomen in the United States chronically suffer from \nsymptoms of endometriosis, which include pelvic pain, \ninfertility, menstrual cycle abnormalities and increased \n3\nKey Words\n f endometriosis\n f steroid receptor \ncoactivator 1 isoform\n f estrogen receptor β\n f bufalin\n f apoptosis\n f pyroptosis\nJournal of Endocrinology  \n(2018) 237, 255–269\n237\nDownloaded from Bioscientifica.com at 06/12/2026 06:18:38PM\nvia free access\n\n\nhttps://doi.org/10.1530/JOE-17-0700\nhttp://joe.endocrinology-journals.org © 2018 Society for Endocrinology\nPublished by Bioscientifica Ltd.\nPrinted in Great Britain\n256\nBufalin suppresses \nendometriosis\nY J Cho, J E Lee et al.237:3\nJournal of \nEndocrinology\nrisk of certain cancers, such as ovarian, breast and skin \ncancers (Vercellini et al. 2014, Farland et al. 2016, 2017, \nBrilhante et al. 2017, Poole et al. 2017).\nDue to the severe chronic morbidity associated with \nthis gynecological disorder, a number of past studies \nhave attempted to identify the distinguishing molecular \nfeatures of the endometriotic lesion with a view to \ndeveloping more effective prognostic, diagnostic and/or \ntreatment strategies in the clinical management of this \ndebilitating disease ( Bedaiwy  et  al. 2017 ). Despite such \nefforts, many of the current clinical treatments are not \nadequately effective at treating this disease and produce \nunacceptable side effects. For example, studies have shown \nthat levels of prostaglandin E2 (PGE2), cyclooxygenase-2 \n(COX-2) and various cytokines are highly elevated in \nendometriotic tissue relative to the normal endometrium, \nsupporting a heightened pro-inflammatory response as \na major component of this disease ( Hirata  et  al. 2011 , \nSacco et al. 2012). Therefore, selective COX-2 inhibitors \nare used as the conventional treatment for this disorder \n(Ebert  et  al. 2005 ). However, COX-2 selective inhibitors \nhave gastrointestinal side effects, even though their side \neffects are much less severe than older non-steroidal anti-\ninflammatory drugs ( Ebert et al. 2005).\nSimilarly, it has been well established that increased \nconcentrations of 17β-estradiol (E2) in endometriotic tissues \narise from locally elevated levels of aromatase along with \nreduced activity of 17 β-hydroxysteroid dehydrogenase-2 \n(Bulun et al. 2010, Lamp et al. 2011). Therefore, along with \nthe anti-inflammatory treatments described earlier, current \nendometriosis treatments also involve suppressing E2 \nlevels through the use of gonadotropin-releasing hormone \nagonists, oral contraceptives, synthetic progestins and/or \naromatase inhibitors (Goenka et al. 2017). However, many \nclinical reports have revealed that these systemic estrogen \ndeficiency therapies cause infertility and confer harmful \nside effects in other estrogen target tissues, such as bone \nand brain.\nBecause of the unacceptable deficiencies cited earlier, \nthere is clearly an urgent need to identify new molecular \nmechanisms that critically underpin the initiation and \nprogression of endometriosis to develop more effective \ntherapeutics that lack the side effects of current treatments. \nInterestingly, our prior study revealed that a steroid receptor \ncoactivator (SRC)-1 isoform that is proteolytically cleaved \nfrom the full-length SRC-1 by matrix metalloproteinase-9 \nand estrogen receptor (ER) β are specifically elevated in \nendometriotic tissues compared to the normal endometrium, \nand this SRC-1 isoform/ER β axis has an essential role \nin endometriosis progression because this axis prevents  \nTNFα-mediated apoptosis and enhances inflammasome-\nmediated inflammatory signaling in endometriotic lesions \nfor their survival ( Han  et  al. 2012 , 2015). Collectively, \nthese recent findings led us to hypothesize that the SRC-1 \nisoform/ERβ axis should be a new molecular therapeutic \ntarget for an alternative endometriosis treatment to \nenhance the specificity of endometriosis treatment and \nreduce the side effects of previous endometriosis treatments. \nOur previous studies defined small molecular inhibitors \n(SMIs), such as bufalin and gossypol, that diminished the \nactivities and protein stabilities of SRCs and suppressed \nthe growth of various cancer cells (Wang et al. 2011, 2014). \nThese observations led us to investigate whether SMIs \nagainst SRCs could be employed to suppress endometriosis \nprogression due to the crucial role of the SRC-1 isoform in \nendometriosis progression. Therefore, we here show that \nbufalin, one of the SMIs against SRCs, represents a new class \nof drugs that could be used to combat endometriosis based \non its antagonistic role against the SRC-1 isoform/ERβ axis \nin endometriotic lesions.\nMaterials and methods\nMice\nMice were housed in a pathogen-free animal facility \nunder a standard 12-h light/12-h darkness cycle and \nwere fed standard rodent chow and water. All animal \nexperimentation was conducted in accordance with \naccepted standards of humane animal care. All animal care \nwas controlled by the ethical regulations approved by the \nInstitutional Animal Care and Use Committee at Baylor \nCollege of Medicine. Five-week-old normal (C57BL/6J) \nmice were purchased from Jackson Laboratory.\nImmortalized human endometrial cells\nPrimary human endometrial stromal cells isolated from \nwomen with/without endometriosis ( Han et  al. 2012 ), \nimmortalized human endometrial stromal cells (IHESCs) \n(Krikun et al. 2004), EMosis-CC/TERT1 (immortalized human \nendometriotic epithelial cells; IHEECs) (Bono et al. 2012) and \nHeLa cells were confirmed by Short Tandem Repeat profiling; \nthese cells were not contaminated with mycoplasma.\nGeneration of a lentivirus expressing the SRC-1 \nisoform\nThe open reading frame of the SRC-1 isoform gene was \ncloned into a pCDH-pCMV vector using BamH1 and XhoI \nDownloaded from Bioscientifica.com at 06/12/2026 06:18:38PM\nvia free access\n\n\nhttps://doi.org/10.1530/JOE-17-0700\nhttp://joe.endocrinology-journals.org © 2018 Society for Endocrinology\nPublished by Bioscientifica Ltd.\nPrinted in Great Britain\n257\nResearch\nY J Cho, J E Lee et al. Bufalin suppresses \nendometriosis\n237:3\nJournal of \nEndocrinology\nrestriction enzymes. Next, 293T cells on a 100-cm tissue \nculture dish were transfected with the pCDH-pCNV-SRC-1 \nisoform and Lenti-X packaging single shot (Clontech \nLaboratories). The virus-containing medium was \ncollected at 48 h after transfection. The lentivirus titer was \ndetermined by Lenti-X GoStix (Clontech Laboratories).\nGeneration of IHESCs and IHEECs that expressed \nthe SRC-1 isoform\nIHEECs and IHESCs were cultured in a 10-cm dish. When \nthe cell confluency reached 70%, 6 mL of new medium \ncontaining 64 μg of polybrene was added, and then 2 mL \nof media containing lentivirus (MOI of approximately \n2) was added. At 2  days after transduction, 2 μg/mL of \npuromycin was added to the media. The puromycin-\nresistant cells were selected, and then the expression \nof the SRC-1 isoform in these cells was determined by \nWestern blot analyses with SRC-1 antibody.\nMTS cell growth assay\nPrimary human endometrial stromal cells isolated \nfrom women with/without endometriosis, IHEECs, \nIHEECs:SRC-1 ISO, IHESCs and IHESCs:SRC-1 ISO were \ninoculated into the wells of 96-well plates (1 × 104 cells/\nwell). The next day, each cell line was treated with serially \ndiluted bufalin (0–800 nM) and vehicle as the control. \nAfter 2  days, 10 μL of MTS reagent was added to each \nwell. MTS-treated plates were incubated for 2 more hours. \nAfter that, the optical density of color in each well was \nmeasured at 490 nm in a microtiter plate reader.\nSurgically induced endometriosis\nEndometriosis in mice was surgically induced under aseptic \nconditions under anesthesia using a modified method \nas described previously ( Cummings & Metcalf 1995 ). \nBriefly, C57BL/6 mice were subjected to ovariectomy \nat six weeks old. After one week, the ovariectomized \nmice were implanted with a sterile, 60-day release pellet \ncontaining 0.36 mg of 17-β estradiol (Innovative Research \nof America, Sarasota, FL, USA). Two days later, one uterine \nhorn from each mouse was isolated under anesthesia. In a \nPetri dish containing warmed DMEM/F-12 supplemented \nwith 100 U/mL penicillin and 100 µg/mL streptomycin, \nthe uterine horns were longitudinally cut with a pair of \nscissors. Next, using a 2-mm dermal biopsy punch, one \nendometrial fragment was isolated and subsequently \nsutured to the mesenteric membrane attached to the \nintestine in the same mouse through a midline incision \n(7-0 braided polypropylene suture). In the case of sham-\ntreated control mice, a suture was performed without \nendometrial tissue fragments. The abdominal incision \nwas then closed with a 5-0 braided polypropylene suture \nin a continuous fashion. On day 21 after endometriosis \nchallenge, the mice were killed, and the endometriotic \nlesions and eutopic endometria were carefully isolated \nfrom the surrounding tissue. Using the formula volume \n(mm3) = 0.52 × width × length × height, the volumes of the \nendometriotic lesions were calculated.\nBufalin treatment of endometriosis-induced mice\nEndometriosis was surgically induced as described \nearlier. Based on a previous study, we injected mice with  \n1 mg/kg of bufalin (Zhang et al. 2014). After endometriotic \nlesions were established (one week after endometriosis \ninduction), the mice were randomly divided into two \ngroups and then subcutaneously administered vehicle (as \nthe control) or 1 mg/kg of bufalin daily for 21 days.\nBufalin treatment of wild-type mice\nFemale C57BL/6J (6 weeks old) were treated with 1 mg/kg  \nof bufalin and vehicle as the control every day for \n21  days. Two weeks before harvesting the uteri, mouse \nestrous cycles were determined using vaginal cytology \n(McLean et al. 2012). At the estrus cycle after 21-day drug \ntreatment, uteri were isolated from mice treated with \nbufalin and vehicle.\nFertility assay following bufalin treatment\nC57BL/6J female mice (8  weeks of age) were treated \ndaily with vehicle and bufalin (1.0 mg/kg) for 21  days  \n(n = 3/group). Afterwards, each female mouse was paired \nwith a wild-type male of proven fertility (1:1). The fertility \nwas assessed by monitoring the litter size over a two-\nmonth period.\nWestern blot analyses\nEndometriotic tissues, human endometrial cells and \ntransfected HeLa cells were washed with PBS solution and \nhomogenized in a buffer containing 10 mM Tris–HCl (pH \n7.4), 150 mM NaCl, 2.5 mM EDTA and 0.5% Nonidet P-40 \n(vol/vol). Cellular debris was removed by centrifugation \nat 12,000 g for 15 min at 4°C. The protein concentration \nwas determined by Bradford’s method using bovine serum \nDownloaded from Bioscientifica.com at 06/12/2026 06:18:38PM\nvia free access\n\n\nhttps://doi.org/10.1530/JOE-17-0700\nhttp://joe.endocrinology-journals.org © 2018 Society for Endocrinology\nPublished by Bioscientifica Ltd.\nPrinted in Great Britain\n258\nBufalin suppresses \nendometriosis\nY J Cho, J E Lee et al.237:3\nJournal of \nEndocrinology\nalbumin as the standard. Samples containing 10 µg total \nproteins were subjected to 10% SDS-polyacrylamide \ngel electrophoresis. The separated proteins were then \ntransferred onto a polyvinylidene difluoride membrane. \nMembranes were blocked overnight with 5% skim milk \n(wt/vol) in phosphate-buffered saline with 0.1% Tween \n20 (vol/vol). Primary antibodies against the following \nproteins were used: SRC-1 (ab10308; Abcam), tubulin  \n(SC-9104; Santa Cruz Biotechnologies), ERα (SC-542; Santa \nCruz Biotechnologies) and chicken anti-ERβ antibody 503 \n(Saji  et  al. 2000 ). Membrane-containing proteins were \nincubated with secondary horseradish peroxidase-tagged \nantibodies (Sigma), and the signals were visualized using \nan enhanced  luminol-based substrate for horseradish \nperoxidase.\nImmunohistochemistry\nImmunostaining was performed with 10% neutral-\nbuffered, formalin-fixed and paraffin-embedded sections \nof mouse tissue, as previously described (Han et al. 2005). \nFor immunostaining, sections were dewaxed, rehydrated \nand boiled for 10 min in 10 mM citrate buffer, pH 6.0. To \nreduce nonspecific binding of antibodies, sections were \nwashed in PBS again and preincubated with 5% BSA in PBS \nfor 1 h at room temperature. Antibodies against PSMD2 \n(A303-854A-T; Bethyl Laboratories), UBA7 (ab133479, \nAbcam), Ki-67 (ab16667; Abcam), cleaved caspase 3 (9664; \nCell Signal), caspase 1 (2225; Cell Signal), active form of \nInterleukin (IL)-1β (521875; Cell Signaling), PERK (5683; \nCell Signal), PDI (3501; Cell signal) and BiP (3177; Cell \nSignal) were used. The specific antigens were visualized \nwith the DAB substrate kit. The immunostaining intensity \nwas quantified using the ImageJ program, which was \ndeveloped by the National Institutes of Health.\nTUNEL assay\nThe TUNEL assay was conducted with a TACS.XL DAB In \nSitu Apoptosis Detection Kit and its protocol (Trevigen, \nInc., Gaithersburg, MD, USA).\nTransfection and luciferase reporter gene assay\nTransfections with plasmids were performed using \nLipofectamine 2000 reagent (Invitrogen) according \nto the manufacturer’s instructions. HeLa cells were \ntransfected with the indicated expression plasmids. For \nthe determination of ERβ transcriptional activity, estradiol \n(10−8 M) was added to cells 24 h following transfection and \nincubated for another 24 h. The cells were harvested, and \nthe luciferase activity was determined and normalized \nagainst the total input protein.\nStatistical analyses\nStatistical analyses were performed by using Windows \nGraphPad Prism 5 (GraphPad Software). The data are \nexpressed as the mean ± s.e.m . Significance was assessed \nusing an independent two-tailed Student’s t-test. A P value \nof less than 0.05 was considered statistically significant. \nN.S., nonspecific.\nResults\nBufalin effectively inhibited the growth of primary \nhuman endometrial stromal cells isolated from \nendometriosis patients and immortalized human \nendometrial cells expressing the SRC-1 isoform\nCompared to levels in the normal endometrium, levels of \nthe SRC-1 isoform are highly elevated in endometriotic \ntissues, and this isoform prevents TNFα-induced apoptosis \nsignaling in endometriotic lesions for their survival ( Han \net al. 2012). Therefore, targeting the SRC-1 isoform should \nreactivate TNF α-induced apoptosis in endometriotic \nlesions, killing them. To inhibit SRC activity, we \npreviously identified SMIs that inhibited the function of \nSRCs. Bufalin, one of the SRC-SMIs, inhibited the intrinsic \ntranscriptional activity of SRC-1 and -3 and degraded \ntheir proteins in various cancer cells to suppress their \ngrowth (Wang et al. 2014). These observations led us to \nexamine whether bufalin might suppress endometriosis \nprogression by inhibiting the function of the SRC-1 \nisoform in endometriotic tissues.\nTo determine whether endometriotic tissues are more \nsensitive to bufalin compared to normal endometrium, \nwe determined the growth pattern of primary human \nendometrial stromal cells isolated from women with/\nwithout endometriosis upon bufalin treatment (Han et al. \n2012). Bufalin effectively inhibited the growth of primary \nhuman endometrial stromal cells from endometriosis \npatients compared to those isolated from women without \nendometriosis ( Fig.  1A ). Therefore, endometriotic cells \nwere more sensitive to bufalin treatment than normal \nendometrial cells.\nIn addition to primary human endometrial cells, we \nemployed IHESCs and IHEECs as human endometrial \nepithelial and stromal cell lines, respectively, because \nIHESCs are karyotypically, morphologically and \nDownloaded from Bioscientifica.com at 06/12/2026 06:18:38PM\nvia free access\n\n\nhttps://doi.org/10.1530/JOE-17-0700\nhttp://joe.endocrinology-journals.org © 2018 Society for Endocrinology\nPublished by Bioscientifica Ltd.\nPrinted in Great Britain\n259\nResearch\nY J Cho, J E Lee et al. Bufalin suppresses \nendometriosis\n237:3\nJournal of \nEndocrinology\nphenotypically similar to the primary parent cells \n(Krikun et  al. 2004 ) and because IHEECs also retain \nthe normal functions and characteristics of the \nprimary cells ( Kyo  et  al. 2003 ). To determine the role \nof the SRC-1 isoform in bufalin-mediated suppression \nof endometriotic cells, we generated recombinant \nIHEECs and IHESCs that stably expressed the SRC-1 \nisoform (IHEECs:SRC-1 ISO and IHESC:SRC-1 ISO) \nusing a lentivirus containing the SRC-1 isoform gene \nexpression unit. The growth of IHESCs:SRC-1 ISO and \nIHEECS:SRC-1 ISO was significantly reduced by bufalin \ntreatment compared to their parental cells ( Fig. 1B  and \nC). Therefore, the overexpression of the SRC-1 isoform \nin human endometrial epithelial and stromal cells \nincreased the sensitivity against bufalin.\nBufalin suppressed the growth of endometriotic \nlesions in mice with endometriosis in vivo\nWe next examined whether bufalin suppresses the \ngrowth of endometriotic lesions in mice with surgically \ninduced endometriosis in vivo because of the essential role \nof the SRC-1 isoform in endometriosis progression. To \naddress this issue, endometriosis was surgically induced \nin C57BL/6J mice using an autotranslation method. \nAfter the establishment of endometriotic lesions in mice \n(at one week after endometriosis induction), mice with \nendometriosis were randomly divided two groups and \nthen injected with bufalin (1.0 mg/kg, daily, n = 4/group) \nor with vehicle for the control (n = 4/group). To determine \nthe effect of bufalin in endometriosis progression, we \nisolated endometriotic lesions from each drug-treated \nmouse with endometriosis and then determined the \nvolume of the endometriotic lesions. Compared to the \nvehicle, bufalin treatment reduced the volume of the \nendometriotic lesions by 14-fold ( P = 0.009) (Fig.  2A ). \nTherefore, bufalin treatment significantly suppressed \nthe growth of endometriotic lesions in mice with \nendometriosis in vivo.\nSince the SRC-1 isoform/ER β axis plays a crucial \nrole in endometriosis progression, we next examined \nwhether bufalin treatment disrupts this SRC-1 isoform/\nERβ functional axis in endometriotic lesions to suppress \ntheir growth. Western blot analysis revealed that \ncompared to the vehicle, bufalin treatment increased \nSRC-1 isoform protein levels by 2.7-fold ( P = 0.005) \nin endometriotic lesions ( Fig.  2B ). To validate this \nbufalin-induced elevation of SRC-1 isoform levels in \nendometriotic lesions, IHESCs:SRC-1 ISO cells were \ntreated with bufalin (0, 5 and 20 nM) for 24 or 48 h. We \nfound that 20 nM bufalin treatment, compared with \nthe vehicle, elevated the SRC-1 isoform protein levels \nby 2.4-fold in IHESCs:SRC-1 ISO with 48-h treatment \n(Fig. 2C ). To define the effect of bufalin on full-length \nSRC-1, endogenous SRC-1 levels in IHESCs treated with \nbufalin were determined by Western blot analyses. In \ncontrast to the results for the SRC-1 isoform, 20 nM \nbufalin treatment, compared with the vehicle, reduced \nthe levels of full-length SRC-1 by 5.0-fold in IHESCs \nwith 48-h treatment ( Fig.  2D ). The bufalin-induced \ndegradation of full-length SRC-1 in various cancer \ncells was also reported in our previous study ( Wang \net al. 2014). Therefore, bufalin increased SRC-1 isoform \nprotein levels but reduced the full-length SRC-1 protein \nlevels in endometriotic tissues.\nIn addition to the SRC-1 isoform, ER β also has an \nessential role in endometriosis progression. Therefore, we \ndetermined whether bufalin also affects the ER β axis in \nendometriotic lesions. In contrast to the results for the \nSRC-1 isoform, however, bufalin treatment, compared to \nCell Viability (%)\nCell Viability (%)\nCell Viability (%)\nBufalin Concentration [Log(nM)] Bufalin Concentration [Log(nM)] Bufalin Concentration [Log(nM)]\nAB C\n0\n20\n40\n60\n80\n100\n120\n0123\nEndo Normal IHEECs:SRC-1ISO IHEECs IHESCs:SRC-1ISO IHESCs\n0.0\n20.0\n40.0\n60.0\n80.0\n100.0\n120.0\n0 123\n0\n20\n40\n60\n80\n100\n120\n0123\nFigure 1\nBufalin inhibited the growth of primary human endometrial stromal cells isolated from endometriosis patients and immortalized human endometrial \ncells expressing the SRC-1 isoform. (A) The growth patterns of primary human endometrial stromal cells isolated from women with (Endo) and without \n(Normal) endometriosis were determined at different concentrations of bufalin (0, 10, 20, 50, 100, 200, 400 and 800 nM) for 48 h of treatment, by using \nthe MTS cell growth assay. (B) The growth patterns of IHEECs expressing the SRC-1 isoform (IHEECs:SRC-1 ISO) and their parental cells were determined \nat different concentrations of bufalin for 48 h, by using the MTS cell growth assay. (C) The growth patterns of IHESCs expressing the SRC-1 isoform \n(IHESCs:SRC-1 ISO) and their parental cells were determined at different concentrations of bufalin for 48 h, by using the MTS cell growth assay. Data are \npresented as the means ± s.e.m .\nDownloaded from Bioscientifica.com at 06/12/2026 06:18:38PM\nvia free access\n\n\nhttps://doi.org/10.1530/JOE-17-0700\nhttp://joe.endocrinology-journals.org © 2018 Society for Endocrinology\nPublished by Bioscientifica Ltd.\nPrinted in Great Britain\n260\nBufalin suppresses \nendometriosis\nY J Cho, J E Lee et al.237:3\nJournal of \nEndocrinology\nthe vehicle, reduced ERβ protein levels by 7.2-fold (P = 0.01) \nin endometriotic lesions (Fig. 2B ). In addition to those of \nendometriotic lesions, ERβ levels in IHESCs:SRC-1 ISO were \nreduced by 5.0-fold by 20 nM bufalin at 48-h treatment \ncompared with the levels after vehicle treatment (Fig. 2C ). \nTherefore, bufalin treatment disrupted the SRC-1 isoform/\nERβ axis by increasing SRC-1 isoform protein levels, \ndecreasing ER β protein levels in endometriotic lesions, \nand then suppressing endometriosis progression.\nBufalin increased the intrinsic transcriptional \nactivity of the SRC-1 isoform but not the ERβ activity\nWe asked whether bufalin also affects the intrinsic \ntranscriptional activity of the SRC-1 isoform in addition \nto protein levels because bufalin inhibits the intrinsic \ntranscriptional activity of full-length SRC-1 ( Wang et al. \n2014). To address this issue, we generated a mammalian \nexpression vector for the chimeric SRC-1 isoform protein \nfused to the Gal4 DNA-binding domain (pBID-SRC-1 \nisoform) and then transiently transfected it into HeLa \ncells along with a pG5 luciferase reporter containing \nGal4 DNA-binding elements. Compared to the empty \nexpression vector control, the SRC-1 isoform had an \nintrinsic transcriptional activity, and its transcriptional \nactivity was elevated by 5 nM bufalin treatment (3-fold, \nP = 0.005) compared to the vehicle ( Fig. 3A ). Collectively, \nbufalin hyperactivated the SRC-1 isoform function in \nendometriotic lesions by stabilizing the SRC-1 isoform \nprotein, stimulating its transcriptional activity.\nWe next determined the effect of bufalin on \nthe intrinsic transcriptional activity of ER β using an  \nERβ/Estrogen Response Element luciferase assay \nsystem in HeLa cells. Compared with the vehicle, \nestrogen treatment significantly enhanced the intrinsic \ntranscriptional activity of ER β ( Fig.  3B ). However, \ncompared to the vehicle, bufalin treatment did not affect \nthe E2-induced transcriptional activity of ER β (Fig. 3B ). \nTherefore, bufalin treatment decreased the protein levels \nof ER β but did not inhibit its transcriptional activity in \nendometriotic lesions.\nVehicle\nA BC D\nBufalin\nVolume of Ectopic Lesions (mm\n3\n)\nVehicle Bufalin\nVehicle Bufalin\nSRC-1\nIsoform\nVehicl\ne\nBufalin\nSRC-1 isoform/Tubulin\n24\n05 20 05 20 nM Bufalin 05 20 05 20 nM Bufalin\n10 .5 0.51 0.40 .213 .1 5.31 1.52 .4\n48 hours 24 48 hours\nSRC-1\nTubulin\nSRC-1\n-Isoform\nP=0.009\nP=0.005\nVehicl\ne\nBufalin\nERβ/Tubulin\nP=0.015\n4\n3\n2\n1\n0\n1.5\n1.0\n0.5\n0\nTubulin \nERβ\nTubulinERβ\nSRC-1\nTubulin \nVehicl\ne\nBufalin\n10 .8 0.81 0.40 .2\nFigure 2\nBufalin suppressed the growth of endometriotic lesions in mice with endometriosis. (A) Endometriotic lesions were isolated from mice with surgically \ninduced endometriosis treated with 1.0 mg/kg bufalin and the vehicle as the control (n = 4 mice for each group). The volume of each endometriotic \nlesion was determined by the formula volume (mm3) = 0.52 × width × length × height. (B) Protein levels of the SRC-1 isoform, ERβ and tubulin were \ndetermined in endometriotic lesions treated with bufalin (1 mg/kg) or vehicle by Western blot analysis (n = 3 mice for each group). (C) IHESCs:SRC-1 ISO \ncells were treated with 5 and 20 nM bufalin for 24 and 48 h, and then protein levels of the SRC-1 isoform, ERβ and tubulin were determined by Western \nblot analysis. (D) IHESCs were treated with 5 and 20 nM bufalin for 24 and 48 h, and then protein levels of the full-length SRC-1 isoform and tubulin were \ndetermined by Western blot analysis. Data are presented as the means ± s.e.m . and P value (Student’s t-test).\npBID-SRC-1 Isoform\n+ pG5 Reporter\nFirefly-Luficerase Activity\nFirefly-Luficerase Activity\nA B ERβ + \nERE-Luciferase Reporter\nSRC-1 Isoform :- ++ +\nBufalin (nM)     :0 02 .5 5.0\nBufalin (nM)     :0 02 .5 5.0\nERβ ++ 2.++\nP=0.005\nN.S.\nE2 (10 nM)      :- ++ +\nFigure 3\nBufalin stimulated the intrinsic transcriptional activity of the SRC-1 \nisoform. (A) HeLa cells were transfected with pBID-SRC-1 isoform and pG5 \nreporter vectors and then treated with 0, 2.5 or 5.0 nM bufalin for 48 h. \nThe luciferase activity in HeLa cells treated with different doses of bufalin \nwas determined to define the intrinsic transcriptional activity of the \nSRC-1 isoform upon bufalin treatment. (B) HeLa cells were transfected \nwith an ERβ expression vector and ERE-luciferase reporter. To stimulate \nERβ activity, HeLa cells were treated with estradiol (10 nM) plus 0, 2.5 or \n5.0 nM bufalin treatment for 48 h. The luciferase activity in HeLa cells \ntreated with different doses of bufalin was determined to define the \nintrinsic transcriptional activity of the ERβ upon bufalin treatment. Data \nare presented as the means ± s.e.m . and P value (Student’s t-test).\nDownloaded from Bioscientifica.com at 06/12/2026 06:18:38PM\nvia free access\n\n\nhttps://doi.org/10.1530/JOE-17-0700\nhttp://joe.endocrinology-journals.org © 2018 Society for Endocrinology\nPublished by Bioscientifica Ltd.\nPrinted in Great Britain\n261\nResearch\nY J Cho, J E Lee et al. Bufalin suppresses \nendometriosis\n237:3\nJournal of \nEndocrinology\nBufalin degraded ERβ protein via the proteasome \n26S subunit, non-ATPase 2 (PSMD2) in \nendometriotic tissues\nHow does bufalin degrade ERβ in endometriotic lesions? To \ndetermine the molecular function of ERβ in endometriosis \nprogression, we isolated the ERβ-containing complex from \nendometriotic lesions using immunoprecipitation and then \nidentified all protein components co-precipitated with ERβ \n(Han et  al. 2015 ). Interestingly, these data revealed that \nseveral proteasome components, such as proteasome 26S \nSubunit, non-ATPase 2 (PSMD2) and ubiquitin-like modifier \nactivating enzyme 7 (UBA7), were specifically co-precipitated \nwith ERβ from endometriotic lesions. This observation led \nus to investigate the potential roles of PSMD2 and UBA7 in \nbufalin-induced ERβ protein degradation in endometriotic \ntissues. To validate this hypothesis, we measured the protein \nlevels of PSMD2 and UBA7 in endometriotic lesions treated \nwith bufalin vs vehicle using immunohistochemistry (IHC). \nBufalin treatment increased the levels of PSMD2 and UBA7 \nin both epithelial and stromal cells from endometriotic \nlesions compared to vehicle-treated endometriotic lesions \n(Fig. 4A  and B).\nWe next examined whether the increased quantities \nof PSMD2 and UBA7 degraded ER β proteins in \nendometriotic lesions. To address this issue, HeLa cells \nwere cotransfected with expression vectors containing  \nERβ plus PSMD2 or ERβ plus UBA7. An empty expression \nvector was also cotransfected into HeLa cells with ERβ \nexpression vector as the control. The overexpression \nof PSMD2 decreased ER β protein levels by 2.5-fold in \nHeLa cells compared with the empty vector control \n(Fig. 4C ). However, the overexpression of UBA7 protein \ndid not degrade ER β protein compared to the empty \nvector control ( Fig. 4C ). Therefore, elevation of PSMD2 \ncould be associated with degradation of ER β protein in \nendometriotic lesions by bufalin treatment. To determine \nthe specificity of PSMD2-mediated ER β degradation, \nmammalian expression vectors for ERα, full-length SRC-1  \nand SRC-1 isoform were also cotransfected into HeLa \ncells along with expression vectors for PSMD2 and UBA7. \nIn contrast with ER β, however, overexpression of both \nPSMD2 and UBA7 did not result in the degradation \nof ER α and SRC-1 isoform proteins ( Fig.  4D  and E). In \ncontrast to the SRC-1 isoform, however, the full-length \nSRC-1 was degraded by PSMD2 but not by UBA7 (Fig. 4F ). \nTherefore, bufalin-mediated degradation of the full-\nlength SRC-1 might be associated with elevation of \nPSMD2 in endometriotic lesions. Collectively, these \nresults imply that bufalin disrupts the SRC-1 isoform/ER β \naxis in endometriotic lesions by degrading ER β protein \nthrough the elevation of PSMD2.\nPSMD2 Levels\nin Epithelum\nVehicleB ufalin\nPSMD2\nUBA7 Levels\nin Epithelium\nP=0.0019\nP=0.0002\nA\nCD EF\nB\n10 .9 0.41 0.90 .9 11 .1 1.21 1.10 .4\nERβ\nControlFLAG-UBA7FLAG-PSMD2\nTubulin\nSRC-1\nIsoform\nERα\nPSMD2 Levels\nin Stroma\nP=0.0001\nUBA7 Levels\nin Stroma\nP=0.0003\nSRC-1\nControlFLAG-UBA7FLAG-PSMD2 ControlFLAG-UBA7FLAG-PSMD2 ControlFLAG-UBA7FLAG-PSMD2\nWB: FLAG\nVehicleB ufalin\nUBA7\nVehicleBufalin Vehicl\ne\nBufalin\nVehicleBufalin Vehicl\ne\nBufalin\nFigure 4\nBufalin degraded ER β in endometriotic lesions via PSMD2. (A and B) Levels of PSMD2 (A) and UBA7 (B) proteins were determined in endometriotic \nlesions isolated from mice with endometriosis treated with bufalin (1 mg/kg) or vehicle by IHC. The expression levels of these proteins in epithelial \ncells and stromal cells of endometriotic lesions were quantified using ImageJ ( n = 3 mice for each group, three independent IHC assays from each \nmouse). (C) To determine ER β degradation by UBA7 or PSMD2, HeLa cells were transfected with the ER β expression vector with the control, UBA7 \nand PSMD2 expression vectors. (D) To define UBA7- or PSMD2-mediated ER α degradation, HeLa cells were transfected with the ER α expression \nvector with the control, UBA7 and PSMD2 expression vectors. (E) To determine whether UBA7 or PSMD2 degrades the SRC-1 isoform, HeLa cells \nwere transfected with the SRC-1 isoform expression vector with the control, UBA7 and PSMD2 expression vectors. (F) To determine whether UBA7 or \nPSMD2 degrades full-length SRC-1, HeLa cells were transfected with the full-length SRC-1 expression vector with the control, UBA7 and PSMD2 \nexpression vectors. At 48th h after transfection, protein levels of ER β, ERα, full-length SRC-1, SRC-1 isoform and tubulin levels were determined by \nWestern blot analyses with their antibodies. The protein levels of UBA7 and PSMD2 were determined by Western blot analyses with FLAG antibody \nbecause the proteins had a FLAG tag in the N-terminal region. Data are presented as the means ± s.e.m . and P value (Student’s t-test).\nDownloaded from Bioscientifica.com at 06/12/2026 06:18:38PM\nvia free access\n\n\nhttps://doi.org/10.1530/JOE-17-0700\nhttp://joe.endocrinology-journals.org © 2018 Society for Endocrinology\nPublished by Bioscientifica Ltd.\nPrinted in Great Britain\n262\nBufalin suppresses \nendometriosis\nY J Cho, J E Lee et al.237:3\nJournal of \nEndocrinology\nBufalin treatment induced apoptosis in epithelial \ncells but reduced the proliferative activity in stromal \ncells from endometriotic lesions\nTo determine the impact of bufalin-induced disruption \nof the SRC-1 isoform/ER β axis in endometriosis \nprogression, we examined the alteration of proliferation \nand apoptosis signaling in endometriotic lesions \ntreated with bufalin compared to the vehicle because \nhyperproliferation and anti-apoptosis are closely \nassociated with endometriosis progression and the \nSRC-1 isoform/ER β axis is involved in dysregulation of \napoptosis and proliferation ( Salmassi  et  al. 2011 , Han \net  al. 2012 , 2015, Pellegrini   et  al. 2012 ). To determine \nthe proliferative activity in endometriotic lesions, Ki-67 \nlevels were determined by IHC ( Fig.  5A ). Proliferation \nin the epithelial compartment of endometriotic lesions \nwas not significantly reduced by bufalin treatment \n(Fig. 5B ). However, bufalin reduced the levels of Ki-67 \nby 62.5% ( P = 0.0032) in the stromal compartment of \nendometriotic lesions compared to the vehicle ( Fig. 5C ). \nTherefore, the bufalin treatment significantly reduced \nthe proliferation of endometrial stromal cells but not \nthat of epithelial cells from endometriotic lesions.\nIn addition to proliferation, we next examined \napoptosis signaling by determining the levels of the \nactive form of caspase 3 ( Fig.  5D ). Bufalin treatment \nincreased the levels of the active form of caspase 3 by \n9.8-fold (P = 0.012) in epithelial cells from endometriotic \nlesions compared to the vehicle ( Fig.  5E ). In contrast \nwith epithelial cells, however, the active form of caspase \n3 was not detected in stromal cells in endometriotic \nlesions treated with bufalin and vehicle ( Fig.  5F ). In \naddition to the active form of caspase3, the TUNEL assay \nalso revealed that compared with the vehicle, the bufalin \ntreatment increased the number of TUNEL-positive cells \nin epithelial cells of the endometriotic lesions ( Fig. 5G  \nand H). In contrast with epithelial cells, however, bufalin, \ncompared with the vehicle, did not elevate TUNEL-\npositive cells in stromal cells of endometriotic lesions \n(Fig.  5I ). Collectively, bufalin treatment is associated \nwith elevation of apoptosis signaling in epithelial cells \nand reduction of proliferation of stromal cells from \nendometriotic lesions.\nFigure 5\nBufalin induced the apoptosis and reduced the \nproliferation in endometriotic lesions. (A, B and \nC) Levels of Ki-67 in endometriotic lesions isolated \nfrom mice with endometriosis treated with \nbufalin (1 mg/kg) and vehicle using IHC (A). The \nlevels of Ki67 in epithelial (B) and stromal cells (C) \nfrom endometriotic lesions in Panel A were \nquantified by ImageJ (n = 5 mice for each group). \n(D, E and F) Levels of the active form of caspase 3 \nprotein (D) in epithelial (E) and stromal cells (F) \nfrom endometriotic lesions treated with bufalin \n(1 mg/kg) or vehicle were determined using IHC \nand then quantified using ImageJ (n = 5 mice for \neach group). (G, H and I) The number of \nTUNEL-positive cells (G) in epithelial (H) and \nstromal cells (I) from endometriotic lesions \ntreated with bufalin (1 mg/kg) or vehicle were \ndetermined using IHC and then quantified using \nImageJ (n = 5 mice for each group). Data are \npresented as the means ± s.e.m . and P value \n(Student’s t-test).\nActive Caspase 3\nVehicle Bufalin\nVehicle Bufalin\nVehicle Bufalin\nKi-67\nP=0.0032\nP=0.012\nN.S.\nTUNEL Assay\nP=0.001 P=N.S.\nPLC (%) of Ki-67\nin Epithelium\nPLC (%) of Ki-67\nin Stroma\nVehicleBufalin Vehicle Bufalin\nVehicleBufalin Vehicl\ne\nBufalin\nVehicleBufalin Vehicl\ne\nBufalin\nPLC (%) of Active\nCaspase 3 in Epithelium\nPLC (%) of Active\nCaspase 3 in Stroma\nPLC (%) of TUNEL\npositive in Epithelium\nPLC (%) of TUNEL\npostive in Stroma\nA BC\nDE F\nGH I\nDownloaded from Bioscientifica.com at 06/12/2026 06:18:38PM\nvia free access\n\n\nhttps://doi.org/10.1530/JOE-17-0700\nhttp://joe.endocrinology-journals.org © 2018 Society for Endocrinology\nPublished by Bioscientifica Ltd.\nPrinted in Great Britain\n263\nResearch\nY J Cho, J E Lee et al. Bufalin suppresses \nendometriosis\n237:3\nJournal of \nEndocrinology\nBufalin treatment induced the pyroptosis signaling \nin stromal cells of endometriotic lesions\nIn addition to antiapoptosis, alteration of inflammatory \nsignaling has an essential role in endometriosis \nprogression. Therefore, we next examined whether \ninflammatory signaling is altered in endometriotic lesions \nby bufalin treatment. Interestingly, bufalin treatment, \ncompared with the vehicle, elevated the active form of \nIL-1β by 5.1-fold ( P = 0.0001) in stromal cells but not in \nepithelial cells ( Fig.  6A , B and C). The elevation of the \nactive form of IL-1 β is associated with the progression \nof pyroptosis ( Ying & Padanilam 2016 ), and caspase I is \nassociated with progression of pyroptosis induced by the \nactive form of IL-1β (Miao et al. 2011). Therefore, we next \ndetermined the levels of caspase I in endometriotic lesions \ntreated with bufalin vs vehicle. In addition to the active \nform of IL-1β, IHC revealed that the levels of caspase 1 were \nelevated by 2.9-fold (P = 0.0015) in stromal cells, but not in \nepithelial cells, from bufalin-treated endometriotic lesions \ncompared to the vehicle-treated lesions ( Fig.  6D , E and \nF). Therefore, bufalin treatment might be associated with \nelevated signaling of the active form of IL-1β by activating \nthe pyroptosis signaling in stromal compartments of \nendometriotic lesions.\nBufalin treatment induced endoplasm reticulum \nstress (ERS) in endometriotic lesions\nPyroptosis elevates the levels of the active form of IL-1 β, \nand then the increased active form of IL-1 β promotes \nERS signaling ( Verma & Datta 2010 , Liu  et  al. 2015 ). \nBased on these observations, we examined the levels of \nERS markers, such as PKR-like ER kinase (PERK), protein \ndisulfide isomerase (PDI) and BiP, in endometriotic lesions \ntreated with bufalin (Oslowski & Urano 2011). Compared \nto the vehicle, bufalin treatment elevated the expression \nlevels of PERK by 9.1-fold ( P = 0.0001) in epithelial cells \nfrom endometriotic lesions ( Fig.  7A  and B). In addition \nto the epithelial compartment, bufalin also increased \nPERK levels in stromal cells from endometriotic lesions by  \n4.1-fold (P = 0.0001) (Fig. 7C ).\nThe PDI levels were also elevated in epithelial \ncells (by 4.2-fold, P = 0.0015) and stromal cells (by 2.9-\nfold, P = 0.0001) in endometriotic lesions treated with \nbufalin compared with the vehicle ( Fig.  7D , E and F). \nLevels of BiP were elevated by 4.6-fold ( P = 0.003) in \nepithelial cells from endometriotic lesions treated with \nbufalin compared with the vehicle ( Fig. 7G  and H). The \nbufalin treatment also elevated BiP levels (by 2.1-fold, \nP = 0.015) in stromal cells from endometriotic lesions by \nbufalin compared with the vehicle ( Fig.  7I ). Therefore, \nbufalin treatment mainly stimulates ERS signaling in \nendometriotic lesions.\nBufalin treatment did not impair normal uterine \nfunction of mice without endometriosis\nOur observations revealed that bufalin treatment elevated \napoptosis, pyroptosis and ERS signaling and reduced the \nproliferation in endometriotic lesions. These observations \nraised the question, what is the effect of bufalin in normal \nuteri? To address this question, normal C57BL/6J mice \nwere treated with bufalin (1 mg/kg for 21  days) and \nFigure 6\nBufalin induced the pyroptosis signaling in \nendometriotic lesions. (A, B and C) Levels of the \nactive form of IL-1β in endometriotic lesions \nisolated from mice with endometriosis treated \nwith bufalin (1 mg/kg) and vehicle using IHC (A). \nThe levels of the active form of IL-1β in epithelial \n(B) and stromal cells (C) from endometriotic \nlesions in Panel A were quantified by ImageJ \n(n = 3 mice for each group, three independent IHC \nassays for each mouse). (D, E and F) Levels of \ncaspase 1 protein (D) in epithelial (E) and stromal \ncells (F) from endometriotic lesions treated with \nbufalin (1 mg/kg) or vehicle were determined \nusing IHC and then quantified using ImageJ (n = 3 \nmice for each group, three independent IHC \nassays for each mouse). Data are presented as the \nmeans ± s.e.m . and P value (Student’s t-test).\nJ\nJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ\nCaspase I\nP=0.0015\nActive IL-1β\nVehicle Bufalin\nVehicle Bufalin\nLevels of Active\nIL-1β in Epithelium\nN.S.\nN.S.\nLevels of Active\nIL-1β in Stroma\nP=0.0001\nVehicl\ne\nBufalin Vehicl\ne\nBufalin\nVehicl\ne\nBufalin Vehicl\ne\nBufalin\nLevels of Caspase I\nin Epithelium\nLevels of Caspase I\nin Stroma\nA BC\nEDF\nDownloaded from Bioscientifica.com at 06/12/2026 06:18:38PM\nvia free access\n\n\nhttps://doi.org/10.1530/JOE-17-0700\nhttp://joe.endocrinology-journals.org © 2018 Society for Endocrinology\nPublished by Bioscientifica Ltd.\nPrinted in Great Britain\n264\nBufalin suppresses \nendometriosis\nY J Cho, J E Lee et al.237:3\nJournal of \nEndocrinology\nvehicle as the control. IHC with Ki-67 antibody revealed \nthat compared to the vehicle, the bufalin treatment did \nnot reduce the proliferation activity in uteri ( Fig. 8A ). In \naddition to proliferation, the level of the active form of \ncaspase 3 was not elevated in uteri by bufalin treatment \ncompared to the vehicle ( Fig.  8B ). IHC with the active \nform of IL-1 β also revealed that the pyroptosis signaling \nwas not elevated in uteri by bufalin treatment compared \nto the vehicle (Fig. 8C ).\nTo further validate the effect of bufalin on normal \nuterine function, we determined the fertility of female \nmice treated with bufalin (1 mg/kg) for 21  days. The \nfemale mouse fertility assay revealed that compared with \nvehicle treatment, bufalin treatment did not reduce the \nreproductive activity in female mice ( Fig.  8D ). In our \nhands, therefore, the 21-day bufalin treatment (1 mg/kg) \ndid not disrupt the fertility of female mice.\nWorking model for bufalin-mediated endometriosis \nsuppression\nBased on our results, we propose a model for bufalin-\ninduced suppression of endometriosis progression (Fig. 9 ). \nBufalin degrades ER β protein by elevating PSMD2 and \nhyperactivates SRC-1 isoform function in endometriotic \nlesions. This disruption of the bufalin-induced SRC-1 \nisoform/ERβ axis is associated with activation of apoptosis \nsignaling in epithelial cells and reduces the proliferation \nof stromal cells. In addition, bufalin treatment stimulates \nthe pyroptosis in stromal cells and then elevates ERS \nsignaling in endometriotic lesions. These multiple cellular \ndysregulations by bufalin treatment in endometriotic \nlesions cause the suppression of endometriosis progression.\nConclusion\nBufalin has been known as a traditional oriental medicine \nand used for cancer treatment because it induces \napoptosis in cancer cells ( Takai et al. 2012). In addition \nto cancer cells, bufalin also induces apoptosis and G0/G1  \ncell cycle arrest in endometriotic stromal cells in vitro  \n(Nasu et al. 2005). However, more detailed studies have \nnot been conducted to evaluate whether bufalin can be \nemployed as an alternative medicine for endometriosis \ntreatment. Bufalin inhibits SRC-1 and SRC-3 functions \nby degrading their protein levels and inhibiting their \nintrinsic transcriptional activities in various cancer cells \nFigure 7\nBufalin induced ERS signaling in endometriotic \nlesions. (A, B and C) Levels of PERK protein (A) in \nepithelial (B) and stromal cells (C) from \nendometriotic lesions treated with bufalin (1 mg/\nkg) or vehicle were determined using IHC and \nthen quantified using ImageJ (n = 3 mice for each \ngroup, three independent IHC assays for each \nmouse). (D, E and F) Levels of PDI protein (D) in \nepithelial (E) and stromal cells (F) from \nendometriotic lesions treated with bufalin  \n(1 mg/kg) or vehicle were determined using IHC \nand then quantified using ImageJ (n = 3 mice for \neach group, three independent IHC assays for \neach mouse). (G, H and I) Levels of BiP protein (G) \nin epithelial (H) and stromal cells (I) from \nendometriotic lesions treated with bufalin  \n(1 mg/kg) or vehicle were determined using IHC \nand then quantified using ImageJ (n = 3 mice for \neach group, three independent IHC assays for \neach mouse). Data are presented as the \nmeans ± s.e.m . and P value (Student’s t-test).\nVehicleB ufalin\nVehicleB ufalin\nVehicleB ufalin\nP=0.0001\nPERK \nP=0.0015\nP=0.003\nA\nP=0.0001\nP=0.015\nP=0.0001\nVehicle Bufalin Vehicle Bufalin\nVehicle Bufalin Vehicle Bufalin\nVehicl\ne\nBufalin Vehicl\ne\nBufalin\nPERK Levels\nin Epithelium\nPDI Levels\nin Stroma\nPDI\nPDI Levels\nin Epithelium\nPERK Levels\nin Stroma\nBiP\nBiP Levels\nin Epithelium\nBiP Levels\nin Stroma\nBC\nDE F\nGH I\nDownloaded from Bioscientifica.com at 06/12/2026 06:18:38PM\nvia free access\n\n\nhttps://doi.org/10.1530/JOE-17-0700\nhttp://joe.endocrinology-journals.org © 2018 Society for Endocrinology\nPublished by Bioscientifica Ltd.\nPrinted in Great Britain\n265\nResearch\nY J Cho, J E Lee et al. Bufalin suppresses \nendometriosis\n237:3\nJournal of \nEndocrinology\n(Wang et al. 2014). However, bufalin hyperactivates the \nSRC-1 isoform function, unlike that of full-length SRC-1,  \nin endometriotic lesions. Since the SRC-1 isoform has \nan essential role in endometriosis progression, further \nactivation of the SRC-1 isoform in endometriotic lesions \nmight stimulate endometriosis progression. However, the \nbufalin-induced hyperactivation of the SRC-1 isoform \nactivity suppressed the growth of endometriotic lesions \ncompared to that of the vehicle. How we can address this \ndiscrepancy? The first explanation is that the molecular \neffects of bufalin on the SRC-1 isoform are quite similar \nto those of another newly discovered small molecule \nnamed MCB613. MCB613 was identified as an SRC small \nmolecule stimulator because treatment with MCB613 \nstimulated the transcriptional activity of SRC and \nmarkedly induced ERS coupled with the generation of \nreactive oxygen species to suppress the growth of cancer \ncells (Wang et al. 2015). Therefore, over-stimulating the \nSRC oncogenic program can be an effective strategy \nto kill cancer cells. In the same context, therefore, \nover-stimulating the endometriosis-promoting SRC-1 \nisoform using bufalin could kill endometriotic lesions \nby promoting ERS induced by pyroptosis, similar to \nMCB613. Therefore, bufalin is an activator of the SRC-1 \nisoform, whereas it acts as an inhibitor against full-length \nSRC-1. The second explanation is that bufalin treatment \ndegrades ER β in endometriotic lesions. Our previous \nstudy revealed that overexpression of the SRC-1 isoform \nin IHEECs prevented TNF α-induced apoptosis because \nIHEECs express ER β ( Han et  al. 2012 ). Similar to SRC-1 \nisoform overexpression, bufalin treatment increased \nthe SRC-1 isoform level and its transcriptional activity \nin endometriotic lesions. However, bufalin treatment \nsuppressed the growth of endometriotic lesions because \nERβ levels were reduced in endometriotic lesions. To \nprevent apoptosis in endometriotic lesions, both the \nFigure 8\nBufalin did not impair the normal uterine \nfunction. (A) Levels of Ki-67 in endometriotic \nlesions isolated from mice treated with bufalin \n(1 mg/kg) and vehicle using IHC. The levels of \nKi-67 in epithelial and stromal cells from uteri in \nPanel A were quantified by ImageJ (n = 5 mice for \neach group). (B) Levels of the active form of \ncaspase 3 protein in epithelial and stromal cells \nfrom uteri treated with bufalin (1 mg/kg) or \nvehicle were determined using IHC and then \nquantified using ImageJ (n = 5 mice for each \ngroup). (C) Levels of the active form of IL-1β in \nepithelial and stromal cells from uteri treated \nwith bufalin (1 mg/kg) or vehicle were determined \nusing IHC and then quantified using ImageJ (n = 5 \nmice for each group). (D) The litter size per \nC57BL/6J female mouse was determined after \n21-day treatment with bufalin (1 mg/kg) and \nvehicle. Data are presented as the means ± s.e.m . \nand P value (Student’s t-test).\nActive Caspase 3\nPLC (%) of Active\nCaspase 3 in Epithelium\nPLC (%) of Ki-67\nin Epithelium\nVehicle Bufalin\nVehicle Bufalin\nVehicle Bufalin\nA\nKi-67Active  IL-1β\nPLC (%) of Ki-67\nin Stromal\nBufalinVehicle BufalinVehicle\nBufalinVehicle BufalinVehicl\ne\nBufalinVehicle BufalinVehicle\nPLC (%) of Active\nCaspase 3 in Stroma\nN.S. N.S.\nN.S. N.S.\nNumber of Female Litters Littersper Female\nVehicle 31 76  ± 1.15\nBufalin(1mg/kg) 31 96  ± 0.57\nLevels of Active\nIL-1β in Epithelium\nN.S.\nLevels of Active\nIL-1β in Stroma\nN.S.\nB\nC\nD\nDownloaded from Bioscientifica.com at 06/12/2026 06:18:38PM\nvia free access\n\n\nhttps://doi.org/10.1530/JOE-17-0700\nhttp://joe.endocrinology-journals.org © 2018 Society for Endocrinology\nPublished by Bioscientifica Ltd.\nPrinted in Great Britain\n266\nBufalin suppresses \nendometriosis\nY J Cho, J E Lee et al.237:3\nJournal of \nEndocrinology\nSRC-1 isoform and ER β axis are required because the \nSRC-1 isoform/ER β complex interacts with the apoptosis \nmachinery in endometriotic lesions ( Han et  al. 2015 ). \nEven though bufalin elevated SRC-1 isoform function \nin endometriotic lesions, it did not prevent apoptosis in \nendometriotic lesions without ER β and then reactivated \napoptosis signaling.\nIn addition to the SRC-1 isoform, bufalin treatment \nalso impaired endometriosis-stimulating ER β signaling \nin endometriotic lesions by degrading ER β protein. ER β \nhas been known as a key driver along with the SRC-1 \nisoform for endometriosis progression by preventing \nTNFβ-induced apoptosis and activating inflammasomes \n(Monsivais et al. 2014 , Han et al. 2015 ). Therefore, ER β \nshould be the best target to suppress endometriosis \nprogression because the deactivation of ER β could \nreactivate TNF α-induced apoptosis signaling only \nin endometriotic lesions to suppress their growth. \nTherefore, bufalin significantly reduced ER β levels in \nendometriotic lesions and then reactivated apoptosis \nsignaling in epithelial cells from endometriotic \nlesions to suppress the endometriosis progression. \nTo degrade ER β protein, bufalin treatment elevated \nlevels of PSMD2 in endometriotic lesions. PSMD2 is \na component of the 19S regulatory component and \nis responsible for substrate recognition and binding. \nInterestingly, the elevation of PSMD2 levels drives \nthe progression of various human diseases. In lung \ncancer, for example, elevated levels of PSMD2 and its \ngene signature are associated with acquisition of the \nmetastatic phenotype and a poor prognosis because \nknockdown of PSMD2 decreases proteasome activity \nand induces growth inhibition and apoptosis in lung \ncancer cell lines ( Matsuyama  et  al. 2011 ). Consistent \nwith the cancer progression, proteasome components \nalso have a crucial role in endometriosis progression \nbecause the proteasome inhibitor bortezomib suppresses \nendometriosis progression in a rat endometriosis model \n(Celik et al. 2008). In contrast with this finding, however, \nthe bufalin-induced elevation of PSMD2 suppresses the \ngrowth of endometriotic lesions. In addition to survival \nfunctions, the proteasome also has an essential role in \ncell death signaling ( Vacca et al. 2007). In endometriotic \ntissues, therefore, PSMD2 specifically recognizes ER β, an \nessential endometriosis driver, and then degrades it to \nsuppress the ERβ-regulated gene signature that is required \nfor endometriosis progression. Interestingly, PSMD2 did \nnot degrade ERα protein. ERα has critical roles in estrogen \ntarget tissues in addition to endometriosis progression, \nand estrogen deficiency affects different tissues, resulting \nin an increase in various diseases such as osteoporosis \nor cardiovascular diseases ( Valera et al. 2015 ). The side \neffects of current endometriosis treatment are partly \ndue to the inhibition of ER α signaling in these estrogen \ntarget tissues. Therefore, the bufalin/PSMD2/ER β \npathway should improve the specificity of endometriosis \ntreatment and reduce side effects of the inhibition of ERα \nsignaling by the current estrogen depletion therapy.\nThe disruption of the SRC-1 isoform/ER β axis \nby bufalin stimulates both pyroptosis and apoptosis \nsignaling in endometriotic lesions. At first, this \nobservation confused us because two different types of \ncell death signaling occurred in endometriotic lesions at \nthe same time. However, IHC analyses revealed that the \ntwo different cell death signaling pathways are detected in \ndifferent cellular compartments of endometriotic lesions. \nFor example, activation of pyroptosis and apoptosis is \ndetected in stromal and epithelial cells of endometriotic \nlesions, respectively. Pyroptosis, or caspase 1-dependent \ncell death, is known as inflammatory cell death signaling \nand is initiated by various pathological stimuli, such \nas stroke, heart attack or cancer ( Bergsbaken  et  al. \n2009). However, the role of pyroptosis in endometriosis \nprogression has not been reported. Here, we revealed that \nbufalin induced pyroptosis in the stromal compartments \nof endometriotic lesions by elevating the active form of \nIL-1β and caspase 1 in stromal cells. IL-1β signaling has been \nknown as a double-edged sword because the elevation of \nthe active form of IL-1 β results in proliferative activity in \nBufalin\nPSMD2\nSRC-1 Isoform\n(Hyperactivation)\nERβ\n(Degradation)\nEpithelium : Apoptosis ERS\nStroma : Proliferation Pyroptosis ERS\nSuppression of Endometriosis\nDisruption of SRC-1 Isoform/ERβ Axis in Endometriotic Lesions\nFigure 9\nThe working model for bufalin-mediated suppression of endometriosis \nprogression. Bufalin hyperactivated the SRC-1 isoform and degraded ERβ \nin endometriotic lesion to disrupt the SRC-1 isoform/ERβ axis. This \ndisruption of the SRC-1 isoform/ERβ axis stimulated apoptosis in epithelial \ncells and reduced proliferation in stromal cells of endometriotic lesions. \nIn addition, bufalin activated pyroptosis in stromal cells and enhanced \nERS in endometriotic lesions. The alteration of these cellular pathways \nshould suppress endometriosis progression by bufalin.\nDownloaded from Bioscientifica.com at 06/12/2026 06:18:38PM\nvia free access\n\n\nhttps://doi.org/10.1530/JOE-17-0700\nhttp://joe.endocrinology-journals.org © 2018 Society for Endocrinology\nPublished by Bioscientifica Ltd.\nPrinted in Great Britain\n267\nResearch\nY J Cho, J E Lee et al. Bufalin suppresses \nendometriosis\n237:3\nJournal of \nEndocrinology\ncertain cases but also induces cell death signaling in other \ncases ( Kolb  et  al. 2014 ). For survival, for example, ER β \nactivated caspase 1 activity in inflammasomes and then \nincreased the active form of IL-1 β levels in endometriotic \nlesions to activate cell adhesion and proliferative activity \nin ectopic lesions ( Han et  al. 2015 ). However, bufalin \ntreatment hyperelevated the levels of the active form \nof IL- β in the stromal compartments of endometriotic \nlesions compared to the vehicle-treated endometriotic \nlesions. Hyperactivating IL-1 β signaling induced ERS, \nresulting in cell death in human pancreatic cells ( Verma \n& Datta 2010 ). Therefore, bufalin treatment elevates \nhyper-releasing active forms of IL-1 β from pyroptotic \nstromal cells and enhances ERS in endometriotic lesions \nto effectively suppress the growth of endometriotic \nlesions along with activation of apoptosis in epithelial \ncells of endometriotic lesions.\nIt appears that bufalin itself may not be an \nideal drug for endometriosis. Bufalin belongs to the \nbufadienolide group, and bufadienolides are known \nfor having deleterious cardiovascular side effects due \nto their inhibition of the transport enzyme Na +/K+-\nadenosine triphosphatase ( Xu  et  al. 2016 ). Despite this \nknown cytotoxic property of bufadienolides, they have \nbeen used to treat cardiovascular and kidney diseases \n(Puschett  et  al. 2010 ). Chronic treatment with a low \ndose of bufadienolides has been proven to be effective in \ncertain instances, causing little to no side effects (Panesar \n1992, Jing et al. 1994). Our study also revealed that 21-day \nbufalin treatment (1 mg/kg) did not impair normal uterine \nfunction and fertility. We know, however, that bufalin has \npotential cardiac ion channel toxicity so it is not an ideal \ncandidate drug with which to go forward. Although the \ncombination therapy of low-dose bufalin and other drugs \ncould be further investigated, this study mainly points to \nthe value of searching in the future for additional new \nchemicals other than bufalin that inhibit SRC function as \npotential therapies for endometriosis.\nDeclaration of interest\nThe authors declare that there is no conflict of interest that could be \nperceived as prejudicing the impartiality of the research reported.\nFunding\nThis work was supported by grants from the US National Eunice Kennedy \nShriver National Institute of Child Health and Human Development \n(NICHD, R01HD082786 and R01HD008188 to B W O), National Institute of \nDiabetes and Digestive and Kidney Diseases (NIDDK, U24 DK097748 Pilot \nGrant to S J H) and Mike Hogg Foundation to S J H.\nAcknowledgment\nS J H led the entire project. S J H and B W O evaluated all data. S J H, Y J C, \nJ L and M J P designed and performed the experiments. S J H, Y J C and J L \nwrote the manuscript. 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