Free Radical Biology and Medicine

β-Sitosterol targets ASS1 for Nrf2 ubiquitin-dependent degradation, inducing ROS-mediated apoptosis via the PTEN/PI3K/AKT signaling pathway in ovarian cancer

The exploration of drugs derived from natural sources holds significant promise in addressing current limitations in ovarian cancer (OC) treatments. While previous studies have highlighted the remarkable anti-cancer properties of the natural compound β-sitosterol (SIT) across various tumors, its specific role in OC treatment remains unexplored. This study aims to investigate the anti-tumor activity of SIT in OC using in vitro and in vivo models, delineate potential mechanisms, and establish a preclinical theoretical foundation for future clinical trials, thus fostering further research. Utilizing network pharmacology, we pinpoint SIT as a promising candidate for OC treatment and predict its potential targets and pathways. Through a series of in vitro and in vivo experiments, we unveil a novel mechanism through which SIT mitigates the malignant biological behaviors of OC cells by modulating redox status. Specifically, SIT selectively targets argininosuccinate synthetase 1 (ASS1), a protein markedly overexpressed in OC tissues and cells. Inhibiting ASS1, SIT enhances the interaction between Nrf2 and Keap1, instigating the ubiquitin-dependent degradation of Nrf2, subsequently diminishing the transcriptional activation of downstream antioxidant genes HO-1 and NQO1. The interruption of the antioxidant program by SIT results in the substantial accumulation of reactive oxygen species (ROS) in OC cells. This, in turn, upregulates PTEN, exerting negative regulation on the phosphorylation activation of AKT. The suppression of AKT signaling disrupted downstream pathways associated with cell cycle, cell survival, apoptosis, migration, and invasion, ultimately culminating in the death of OC cells. Our research uncovers new targets and mechanisms of SIT against OC, contributing to the existing knowledge on the anti-tumor effects of natural products in the context of OC. Additionally, this research unveils a novel role of ASS1 in regulating the Nrf2-mediated antioxidant program and governing redox homeostasis in OC, providing a deeper understanding of this complex disease.

The opposite role of lactate dehydrogenase a (LDHA) in cervical cancer under energy stress conditions

Due to insufficient and defective vascularization, the tumor microenvironment is often nutrient-depleted. LDHA has been demonstrated to play a tumor-promoting role by facilitating the glycolytic process. However, whether and how LDHA regulates cell survival in the nutrient-deficient tumor microenvironment is still unclear. Here, we sought to investigate the role and mechanism of LDHA in regulating cell survival and proliferation under energy stress conditions. Our results showed that the aerobic glycolysis levels, cell survival and proliferation of cervical cancer cells decreased significantly after inhibition of LDHA under normal culture condition while LDHA deficiency greatly inhibited glucose starvation-induced ferroptosis and promoted cell proliferation and tumor formation under energy stress conditions. Mechanistic studies suggested that glucose metabolism shifted from aerobic glycolysis to mitochondrial OXPHOS under energy stress conditions and LDHA knockdown increased accumulation of pyruvate in the cytosol, which entered the mitochondria and upregulated the level of oxaloacetate by phosphoenolpyruvate carboxylase (PC). Importantly, the increase in oxaloacetate production after absence of LDHA remarkably activated AMP-activated protein kinase (AMPK), which increased mitochondrial biogenesis and mitophagy, promoted mitochondrial homeostasis, thereby decreasing ROS level. Moreover, repression of lipogenesis by activation of AMPK led to elevated levels of reduced nicotinamide adenine dinucleotide phosphate (NADPH), which effectively resisted ROS-induced cell ferroptosis and enhanced cell survival under energy stress conditions. These results suggested that LDHA played an opposing role in survival and proliferation of cervical cancer cells under energy stress conditions, and inhibition of LDHA may not be a suitable treatment strategy for cervical cancer.

Apomorphine facilitates loss of respiratory chain activity in human epithelial ovarian cancer and inhibits angiogenesis in vivo

Apomorphine, a therapeutic agent for neurological diseases, is structurally similar to dopamine, and thereby holds potential in cancer therapy. However, there are no reports regarding its anti-cancer effects on human epithelial ovarian cancers (EOCs); therefore, we aimed to elucidate the mechanism underlying its action after drug repositioning. Apomorphine inhibited the proliferation of ES2 and OV90 EOC cells by inducing caspase activation and mitochondrion-associated apoptosis; it also promoted endoplasmic reticulum stress and mitochondrial dysfunction through mitochondrial membrane potential depolarization and mitochondrial calcium overload. Moreover, following apomorphine treatment, we noted the loss of respiratory chain activity by reduction of oxidative phosphorylation and energy-production shift in EOC cells. Further, we verified the anti-angiogenic capacity of apomorphine using fli:eGFP transgenic zebrafish. As a preclinical assessment, we demonstrated the synergistic anti-cancer effects of apomorphine and paclitaxel combination.

Ailanthone increases oxidative stress in CDDP-resistant ovarian and bladder cancer cells by inhibiting of Nrf2 and YAP expression through a post-translational mechanism

Chemoresistance represents one of the main obstacles in treating several types of cancer, including bladder and ovarian cancers, and it is characterized by an increase of cellular antioxidant potential. Nrf2 and YAP proteins play an important role in increasing chemoresistance and in inducing antioxidant enzymes. It has been reported that Ailanthone (Aila), a compound extracted from the Ailanthus Altissima, has an anticancer activity toward several cancer cell lines, including chemoresistant cell lines. We have examined the effect of Aila on proliferation, migration and expression of Nrf2 and YAP proteins in A2780 (CDDP-sensitive) and A2780/CP70 (CDDP-resistant) ovarian cancer cells. Furthermore, to clarify the mechanism of Aila action we extended our studies to sensitive and CDDP-resistant 253J-BV bladder cancer cells, which have been used in a previous study on the effect of Aila. Results demonstrated that Aila exerted an inhibitory effect on growth and colony formation of sensitive and CDDP-resistant ovarian cancer cells and reduced oriented cell migration with higher effectiveness in CDDP resistant cells. Moreover, Aila strongly reduced Nrf2 and YAP protein expression and reduced the expression of the Nrf2 target GSTA4, and the YAP/TEAD target survivin. In CDDP-resistant ovarian and bladder cancer cells the intracellular oxidative stress level was lower with respect to the sensitive cells. Moreover, Aila treatment further reduced the superoxide anion content of CDDP-resistant cells in correlation with the reduction of Nrf2 and YAP proteins. However, Aila treatment increased Nrf2 and YAP mRNA expression in all cancer cell lines. The inhibition of proteolysis by MG132, a proteasoma inhibitor, restored Nrf2 and YAP protein expressions, suggesting that the Aila effect was at post-translational level. In accordance with this observation, we found an increase of the Nrf2 inhibitor Keap1, a reduction of p62/SQSTM1, a Nrf2 target which leads Keap1 protein to the autophagic degradation, and a reduction of P-YAP. Moreover, UCHL1 deubiquitinase expression, which was increased in bladder and ovarian resistant cells, was down-regulated by Aila treatment. In conclusion we demonstrated that Aila can reduce proliferation and migration of cancer cells through a mechanism involving a post translational reduction of Nrf2 and YAP proteins which, in turn, entailed an increase of oxidative stress particularly in the chemoresistant lines.

Efficient iron utilization compensates for loss of extracellular matrix of ovarian cancer spheroids

Metastasis is the major cause of death in women with advanced ovarian cancer. Epithelial ovarian cancer cells can dissociate directly from extracellular matrix (ECM) and form spheroids to spread through the peritoneal cavity. Loss of ECM hinders the survival of ECM-detached epithelial cells. It is still largely unknown how ovarian cancer spheroids maintain their viability after loss of ECM. We find that spheroids derived either from ovarian cancer ascites or cell lines are iron-replete. In accordance with iron-replete condition, proteins involved in iron uptake, transport and storage including divalent metal ion transporter 1 (DMT1), transferrin receptor 1 (TFR1), ferritin, poly(rC)-binding proteins 1 and 2 (PCBP1 and 2) and nuclear factor E2-related factor 2 (NRF2) all increase in ovarian cancer spheroids. Genes linking iron homeostasis and lipid metabolism including stearoyl coenzyme A desaturase 1 (SCD1) are up-regulated in ovarian cancer spheroids. The product of SCD1 oleic acid can restore the viability of ovarian cancer spheroids inhibited by deprivation of iron. Extracellular signal-regulated kinase (ERK) activation contributes to autophagy activation in ovarian cancer spheroids. Impairment of autophagy by U0126 or Olaparib results in lysosomal iron accumulation and decrease of the cytosolic labile iron pool, leading to reduction of SCD1, lipid level and cell viability. Combination of U0126 and Olaparib has synergistic cytotoxicity toward ovarian cancer spheroids. Our findings reveal that ovarian cancer spheroids develop efficient iron utilization system to survive. Targeting iron utilization in ovarian cancer spheroids may have the potential to become new treatment strategies for ovarian cancer metastasis.

Harnessing the synergy of nanosecond high-power microwave pulses and cisplatin to increase the induction of apoptosis in cancer cells through the activation of ATR/ATM and intrinsic pathways

High-adhesion ovarian cancer cell resistance to ferroptosis: The activation of NRF2/FSP1 pathway by junctional adhesion molecule JAM3

Ovarian cancer remains a significant challenge due to the lack of effective treatment and the resistance to conventional therapies. Ferroptosis, a form of regulated cell death characterized by iron-depend and lipid peroxidation, has emerged as a potential therapeutic target in cancer. Ovarian cancer has been reported to exert an "iron addiction" phenotype which makes it is susceptible to ferroptosis inducers. However, we found here that high-adhesion ovarian cancer cells were resistant to ferroptosis. Mechanistically, by PCR array, we identified junctional adhesion molecule 3 (JAM3) as a key mediator of ferroptosis resistance in high-adhesion ovarian cancer cells. Knockdowning and blocking JAM3 sensitized cancer cells to ferroptosis inducers RSL3 and erastin, while JAM3 overexpression conferred resistance to these agents. In addition, JAM3 also promoted ovarian cancer cells resistance to chemotherapeutic agent cisplatin in vitro and in vivo by inhibiting ferroptosis. Furthermore, we demonstrated that JAM3 promoted ferroptosis resistance through NRF2-induced upregulation of FSP1, a critical suppressor of lipid peroxidation. Inhibition of the NRF2/FSP1 pathway eliminated high-adhesion, JAM3 overexpressed ovarian cancer cells resistance to ferroptosis, and decreased cancer cells resistance to cisplatin. Moreover, JAM3 high expression was associated with poor prognosis in patients with ovarian cancer. Altogether, this study provided novel insights into the molecular mechanisms underlying ferroptosis resistance and identify JAM3 as a potential therapeutic target for combating drug resistance in ovarian cancer.

Sodium molybdate inhibits the growth of ovarian cancer cells via inducing both ferroptosis and apoptosis

Ovarian cancer has the most mortality of all gynecologic malignancies. High-grade serous ovarian carcinoma (HGSOC) is the most common and deadly type of ovarian cancer. Tumor recurrence occurs due to the emergence of chemotherapy resistance. Thus, searching for new therapeutic strategies is essential for the management of ovarian cancer. Deregulation of iron metabolism can be used by ovarian cancer cells to survive, proliferate and metastasize. Here we report that sodium molybdate, a soluble molybdenum (Mo) compound, induces the elevation of the labile iron pool (LIP) in ovarian cancer cells, correlated with the down-regulation of genes involved in extracellular matrix organization. Sodium molybdate also induces depletion of glutathione (GSH) through mediating the production of nitric oxide (NO). Elevation of LIP and depletion of GSH promote the ferroptosis of ovarian cancer cells. Meanwhile, nitric oxide induces mitochondrial damage through inhibiting mitochondrial aconitase activity, ATP production, and mitochondrial membrane potential, leading to apoptosis of ovarian cancer cells. In vivo study shows that sodium molybdate reduces tumor burden in nude mice. Xenografts treated with sodium molybdate are characterized by obvious iron accumulation, increased expression of the iron storage protein ferritin, and lipid peroxide product 4-hydroxynonenal. In addition, an elevated percentage of apoptotic cells is observed in xenografts treated with sodium molybdate. Taken together, these results demonstrate that sodium molybdate can induce both ferroptosis and apoptosis of ovarian cancer cells, making it a potential therapeutic candidate for ovarian cancer.

An assessment of serum oxidative stress and antioxidant parameters in patients undergoing treatment for cervical cancer

Oxidative stress and antioxidants are involved in all aspects of cervical cancer. The present study evaluated serum levels of oxidative stress and antioxidant biomarkers in cervical cancer patients and healthy controls. Moreover, the effect of Concurrent chemoradiotherapy (CCRT) on these biomarkers and their association with treatment outcome was investigated. This study included ninety-seven cervical cancer patients and thirty controls. Three oxidative stress parameters (8-hydroxy-2-deoxyguanosine, Protein Carbonyl, and Malondialdehyde) and four antioxidant parameters (Superoxide Dismutase, Catalase, Glutathione Peroxidase, and Total Antioxidant Status) were measured. The analysis was conducted using repeated measures ANOVA for comparing among the phases (before, during, and follow-up) of treatment. The control group was compared using the Dunnet test. Logistic regression analysis was also conducted between oxidative stress and antioxidant parameters to study their association. Significant rises in oxidative damage markers were observed in cervical cancer patients of all stages, compared to controls. There was a further increase in oxidative stress markers during CCRT among complete responders. However, among non-responders, the oxidative stress biomarkers like Protein Carbonyl and Malondialdehyde were unaltered during CCRT. Simultaneously, there was a significant decrease in antioxidant parameters in cervical cancer patients of all stages compared to controls. During CCRT, antioxidant levels continuously depleted among complete responders. Nevertheless, in non-responders, antioxidant parameters like Superoxide Dismutase and Total Antioxidant Status were consistent. The oxidative stress markers and antioxidant parameters normalized among complete responders at six months follow up. While in non-responders, the normalization of these parameters was not observed. Our results indicate that increased oxidative stress and diminished antioxidants among patients were associated with carcinoma cervix. Induced oxidative stress and decreased antioxidant parameters during CCRT among the complete responders show the treatment's efficacy. Oxidant-antioxidant profile merits investigation as markers of diagnosis, treatment response, survival, and recurrence in extensive prospective studies.

A comparative analysis indicates SLC7A11 expression regulate the prognostic value of KEAP1-NFE2L2-CUL3 mutations in human uterine corpus endometrial carcinoma

Uterine corpus endometrial cancer (UCEC) is a third most common malignancy in women with a poor prognosis in advanced stages. In this study, we performed an integrated comparative analysis of exome and transcriptome data from The Cancer Genome Atlas (TCGA) of Lung Adenocarcinoma (LUAD), and UCEC patients. Our multi-omics analysis shows that the UCEC patients carrying mutations in the KEAP1-NFE2L2-CUL3 genes were associated with better progression-free survival (PFS), whereas the KEAP1-NFE2L2-CUL3 mutation in LUAD showed poor outcomes. Functional annotations and correlative expression studies show that genes, particularly GCLC and GCLM related to glutathione synthesis are expressed at lower levels in the KEAP1-NFE2L2-CUL3 mutant UCEC compared to LUAD. This events result in glutathione deficiency and it may compromise to combat intracellular reactive oxygen species (ROS). However, the expression of genes involved in the glutathione recycling process was not affected. On the other hand, cellular import of cystine is high due to increased SLC7A11 expression in UCEC. Because glutathione synthesis is impaired, the unconverted cysteine accumulates in cells, leading to di-sulfite stress. Apart from NRF2, ARID1A is one of the positive regulators of SLC7A11. In support, UCEC patients with co-occurrence of KEAP1-NFE2L2-CUL3 and ARID1A mutation shows significantly decreased PFS with decline of SLC7A11 expression as compared to patients carrying only KEAP1-NFE2L2-CUL3 mutation. Thus, we hypothesize that the KEAP1-NFE2L2-CUL3 mutation in UCEC leads to uncontrollable ROS with di-sulfite stress, reflecting a favorable clinical outcome.

Turning off the ferroptosis switch: ACAA1-Driven PI3K/AKT/Nrf2 signaling as a novel driver of endometrial cancer progression

Endometrial carcinoma (EC) is a prevalent gynecologic malignancy with rising global incidence. Dysregulated lipid metabolism promotes EC progression through estrogen synthesis, metabolic reprogramming, and tumor microenvironment remodeling. Ferroptosis, an iron-dependent cell death driven by lipid peroxidation, represents a potential therapeutic strategy, yet its resistance mechanisms in EC remain unclear. We identify Acetyl-CoA Acetyltransferase 1 (ACAA1), a key enzyme in mitochondrial fatty acid β-oxidation, as an oncogenic factor in EC. We demonstrate that ACAA1 is significantly upregulated in EC tissues via bioinformatic analysis and clinical samples. Functionally, ACAA1 overexpression enhances tumor cell proliferation, migration, energy metabolism, and lipid droplet synthesis in vitro, while accelerating tumor growth in vivo in xenograft models. Mechanistically, ACAA1 activates the PI3K/AKT pathway, leading to nuclear translocation of the transcription factor Nrf2. This ACAA1/PI3K/AKT/Nrf2 axis suppresses ferroptosis by regulating redox homeostasis and lipid peroxidation, thereby promoting EC progression. Our findings reveal ACAA1 as a novel regulator of ferroptosis resistance and tumorigenesis in EC, highlighting its potential as a promising therapeutic target for EC treatment.

Mutant p53 disrupts antioxidant defense in fallopian tube epithelium via GSTAs suppression: A pathway to serous tubal carcinogenesis

High grade serous ovarian cancer (HGSOC) is the most common and aggressive type of epithelial ovarian cancer. The fimbria of the fallopian tube is the likely site of origin based on the presence of distinct precancerous lesions with TP53 signatures known clinically as serous tubal intraepithelial carcinoma (STICs) detected in this region in individuals at genetically high risk or with HGSOC. Previously we identified that matched fallopian tube epithelia (FTE) from fimbria and ampulla of normal fallopian tubes from premenopausal women exhibit differential expression of genes associated with antioxidant and inflammatory pathways. One gene, glutathione S-transferase 2 (GSTA2), showed both higher expression in the fimbria and in the follicular phase (pre-ovulation) compared to the ampulla, suggesting that GSTA2 expression may regulate reactive oxygen homeostasis in these cells in response to ovulation-related stress. Here, to understand how preneoplastic genomic alterations influence regulation of oxidative stress, FTE cells were isolated from healthy tissue and introduced with p53 mutations, from which expression and function of GSTA2 and other antioxidant enzymes were investigated. Mutant p53 downregulated the expression of GSTA2 and subsequently increased DNA damage. The combination of p53 mutation and dysregulated oxidative response likely promotes the genomic instability that initially drives the transformation to high grade serous ovarian carcinoma.

MARCH5-mediated downregulation of ACC2 promotes fatty acid oxidation and tumor progression in ovarian cancer

Lipid metabolic reprogramming has been recognized as a hallmark of human cancer. Acetyl-CoA Carboxylases (ACCs) are key rate-limiting enzymes involved in fatty acid metabolism regulation by catalyzing the carboxylation of acetyl-CoA to malonyl-CoA. Previously, most studies focused on the role of ACC1 in fatty acid metabolism in cancer, while the function of ACC2 remains largely uncharacterized in human cancers, especially in ovarian cancer (OC). Here, we show that ACC2 was significantly downregulated in cancerous tissue of OC, and the downregulation of ACC2 is closely associated with lager tumor size, metastases and worse prognosis in OC patients. Downregulation of ACC2 promoted proliferation and metastasis of OC both in vitro and in vivo by enhancing FAO. Notably, mitochondria-associated ubiquitin ligase (MARCH5) was identified to interact with and downregulate ACC2 by ubiquitination and degradation in OC. Moreover, ACC2 downregulation-enhanced FAO contributed to the progression of OC promoted by MARCH5. In conclusion, our findings demonstrate that MARCH5-mediated downregulation of ACC2 promotes FAO and tumorigenesis in OC, suggesting MARCH5-ACC2 axis as a potent candidate for the treatment and prevention of OC.

SLC27A4-mediated selective uptake of mono-unsaturated fatty acids promotes ferroptosis defense in hepatocellular carcinoma

Aberrant lipid metabolism mediated by the selective transport of fatty acids plays vital roles in cancer initiation, progression, and therapeutic failure. However, the biological function and clinical significance of abnormal fatty acid transporters in human cancer remain unclear. In the present study, we reported that solute carrier family 27 member 4 (SLC27A4) is significantly overexpressed in 21 types of human cancer, especially in the fatty acids-enriched microenvironment surrounding hepatocellular carcinoma (HCC), breast cancer, and ovarian cancer. Upregulated SLC27A4 expression correlated with shorter overall and relapse-free survival of patients with HCC, breast cancer, or ovarian cancer. Lipidomic analysis revealed that overexpression of SLC27A4 significantly promoted the selective uptake of mono-unsaturated fatty acids (MUFAs), which induced a high level of MUFA-containing phosphatidylcholine and phosphatidylethanolamine in HCC cells, consequently resulting in resistance to lipid peroxidation and ferroptosis. Importantly, silencing SLC27A4 significantly promoted the sensitivity of HCC to sorafenib treatment, both in vitro and in vivo. Our findings revealed a plausible role for SLC27A4 in ferroptosis defense via lipid remodeling, which might represent an attractive therapeutic target to increase the effectiveness of sorafenib treatment in HCC.

ICAT drives lactylation of tumor-associated macrophages via the c-Myc-ENO1 axis to promote cervical cancer progression

The effectiveness of immunotherapy in cervical cancer (CC) is profoundly influenced by the tumor microenvironment (TME), where a high infiltration of M2-type tumor-associated macrophages (TAMs) correlates with poor therapeutic responses. Therefore, understanding the molecular mechanisms driving M2-type TAM polarization and identifying novel therapeutic targets are essential for enhancing immunotherapy outcomes in CC. In this study, ICAT was revealed to be significantly upregulated in CC, correlating with poor prognosis. Mechanistically, ICAT facilitated the nuclear translocation of c-Myc, enhancing ENO1 transcription, thereby promoting glycolytic activity and lactate accumulation in the TME. Tumor-derived lactate induced H3K18 lactylation in TAMs, which in turn activated ARG1 expression, driving M2 polarization and establishing an immunosuppressive microenvironment that supports immune evasion. In summary, this study demonstrates that ICAT, by regulating the c-Myc-ENO1 axis, mediates the interaction between tumor cells and macrophages, thereby reshaping the TME and promoting the migration, invasion, and glycolysis of CC. These findings demonstrate that ICAT represents a potential therapeutic target for the treatment of CC.

Von Willebrand factor promotes radiation-induced intestinal injury (RIII) development and its cleavage enzyme rhADAMTS13 protects against RIII by reducing inflammation and oxidative stress

Patients with abdominopelvic cancer undergoing radiotherapy commonly develop radiation-induced intestinal injury (RIII); however, its underlying pathogenesis remains elusive. The von Willebrand factor (vWF)/a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) axis has been implicated in thrombosis, inflammation, and oxidative stress. However, its role in RIII remains unclear. In this study, the effect of radiation on vWF and ADAMTS13 expression was firstly evaluated in patients with cervical cancer undergoing radiotherapy and C57BL/6J mice exposed to different doses of total abdominal irradiation. Then, mice with the specific deletion of vWF in the platelets and endothelium were established to demonstrate the contribution of vWF to RIII. Additionally, the radioprotective effect of recombinant human (rh) ADAMTS13 against RIII was assessed. Results showed that both the patients with cervical cancer undergoing radiotherapy and RIII mouse model exhibited increased vWF levels and decreased ADAMTS13 levels. The knockout of platelet- and endothelium-derived vWF rectified the vWF/ADAMTS13 axis imbalance; improved intestinal structural damage; increased crypt epithelial cell proliferation; and reduced radiation-induced apoptosis, inflammation, and oxidative stress, thereby alleviating RIII. Administration of rhADAMTS13 could equally alleviate RIII. Our results demonstrated that abdominal irradiation affected the balance of the vWF/ADAMTS13 axis. vWF exerted a deleterious role and ADAMTS13 exhibited a protective role in RIII progression. rhADAMTS13 has the potential to be developed into a radioprotective agent.

Plasma-activated medium inhibits cancer stem cell-like properties and exhibits a synergistic effect in combination with cisplatin in ovarian cancer

Ovarian cancer stem-like cells (CSCs) have been implicated in tumor recurrence, metastasis, and drug resistance. Accumulating evidence has demonstrated the antitumor effect of plasma-activated medium (PAM) in various carcinomas, including ovarian cancer. Thus, PAM represents a novel onco-therapeutic strategy. However, its impact on ovarian CSCs is unclear. Here, we show that ovarian CSCs resistant to high-dose conventional chemotherapeutic agents used for ovarian cancer treatment exhibited dose-dependent sensitivity to PAM. In addition, PAM treatment reduced the expression of stem cell markers and sphere formation, along with the aldehyde dehydrogenase- or CD133-positive cell population. We further investigated the effect of PAM in combination with other chemotherapeutics on ovarian CSCs in vitro. PAM exhibited synergistic cytotoxicity with cisplatin (CDDP) but not with paclitaxel and doxorubicin. In a peritoneal metastasis xenograft model established via intraperitoneal spheroid injection, PAM intraperitoneal therapy significantly suppressed peritoneal carcinomatosis (tumor size and number), with a more significant decrease observed due to the combined effects of PAM and CDDP with no side effects. Taken together, our results indicate that PAM inhibits ovarian CSC traits and exhibits synergetic cytotoxicity with CDDP, demonstrating PAM as a promising intraparietal chemotherapy for enhancing antitumor efficacy and reducing side effects.