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WO2023246940A1 - Procédé de traitement d'un cancer par suppression de la croissance de cellules souches cancéreuses, et régulation à la baisse de la voie wnt - Google Patents

Procédé de traitement d'un cancer par suppression de la croissance de cellules souches cancéreuses, et régulation à la baisse de la voie wnt Download PDF

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WO2023246940A1
WO2023246940A1 PCT/CN2023/102178 CN2023102178W WO2023246940A1 WO 2023246940 A1 WO2023246940 A1 WO 2023246940A1 CN 2023102178 W CN2023102178 W CN 2023102178W WO 2023246940 A1 WO2023246940 A1 WO 2023246940A1
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cancer
dhea
cells
combination
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Chi-Ying Huang
Peter Mu-Hsin CHANG
Tai-Shan CHENG
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • A61K31/568—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone
    • A61K31/5685—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone having an oxo group in position 17, e.g. androsterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • the present invention relates to a method for treating a cancer through suppressing the growth of the cancer stem-like cells and the downregulation of WNT pathway.
  • Cancer stem cells are a subpopulation of cancer cells that possess self-renewal capacity and pluripotency. CSCs are involved in tumor development, cell proliferation, and metastasis, and are the key “seeds” for tumor initiation, metastasis, and resistance to chemo-and radiotherapies [1, 3–5] . These processes are regulated by several key transcription factors involved in cancer stemness and sphere formation, such as OCT4, Nanog, SOX2, KLF4, and MYC. Additionally, many signaling pathways, such as the WNT and Notch pathways, also contribute to the development of cancer stemness [6–10] .
  • the WNT signaling pathway involves in cell proliferation, survival, and progression, and influences the self-renewal of stem cells under physiological and pathological conditions [11, 12] .
  • unphosphorylated ⁇ -catenin translocates into the nucleus and subsequently triggers TCF/LEF-mediated transcription of downstream genes, such as CCND1, MYC, and CD44.
  • Dysregulation of the WNT/ ⁇ -catenin signaling pathway is strongly associated with tumorigenesis and progression by maintaining cancer stemness [13] .
  • Recent studies have focused on the therapeutic potential of agents targeting WNT signaling for cancer treatment in mono-or combination therapy [14] .
  • Irinotecan is a topoisomerase I inhibitor that has anticancer activity in solid tumors, such as metastatic colorectal and lung cancer [15–17] .
  • IRN showed some clinical benefit in recurrent or metastatic HNSCC (R/M HNSCC) [18–20] .
  • IRN is a prodrug that is converted into the active metabolite SN-38 by carboxylesterase (CES) 1 or 2 [17] .
  • CES1 was found to be a poor prognostic marker for HNSCC in TCGA HNSCC cohort [21] . It was upregulated in patients with poor prognosis and represented a good therapeutic target for IRN therapy [16, 17, 21] .
  • IRN mono-and combination therapies with other chemotherapeutic agents have been shown to improve the treatment response in cancer patients [19, 20, 22] .
  • survival rate is referred to the percentage of people in a study or treatment group who are still alive for a certain period of time after they were diagnosed with or started treatment for a disease, such as cancer.
  • the combination of IRN with cisplatin showed synergistic anticancer effect in a phase II trial [19] and the cisplatin/tegafur-uracil (UFUR) /irinotecan triple combination therapy demonstrated a moderate response in patients with R/M HNSCC [18] . Toxicity to patients was tolerable, and the quality of life of the patients improved [18] .
  • IRN also induces side effects, such as diarrhea and neutropenia, which can be resolved by optimizing the treatment dosage or increasing the target specificity.
  • DHEA Dehydroepiandrosterone
  • DHEA-S DHEA sulfate
  • DHEA and DHEA-S are both the most abundant steroids in the human serum and are precursors of sex hormones, such as estrogen and androgen.
  • DHEA has been reported to have several beneficial effects such as anti-obesity, hypoglycemia, anti-atherosclerosis, anti-aging, and memory-enhancing effects [27–29] .
  • DHEA has anticancer effects in vitro and in vivo in several cancer types, including breast [30–32] , hepatoma [27] , myeloma [33] , leukemia [34] , colon adenocarcinoma [35] , pancreatic cancer [36] and cervical cancer [37] .
  • breast cancer DHEA inhibited cell proliferation and metastatic processes, such as migration, invasion, and epithelial mesenchymal transition (EMT) , and decreased spheroid size [30–32] .
  • EMT epithelial mesenchymal transition
  • DHEA suppressed stem cell gene expression [38] which suggests that DHEA may have the ability to suppress CSCs.
  • DHEA Dehydroepiandrosterone
  • a chemotherapeutic drug has anti-tumor and anti-stemness efficacy, particularly in treatment of a cancer, including a head and neck cancer such as Head and neck squamous cell carcinomas (HNSCC) , a lung cancer such as non-small-cell lung cancer (NSCLC) and a colorectal cancer (CRC) , or a drug-resistant cancer.
  • HNSCC Head and neck squamous cell carcinomas
  • NSCLC non-small-cell lung cancer
  • CRC colorectal cancer
  • the present invention provides use of dehydroepiandrosterone (DHEA) or its derivatives or metabolites for manufacturing a medicament for treating an anti-cancer drug-resistant cancer with high expression of CES1/2 cells; wherein the cancer is selected from the group consisting of a head and neck cancer, a lung cancer and a colorectal cancer (CRC) .
  • DHEA dehydroepiandrosterone
  • CRC colorectal cancer
  • DHEA is more sensitive to KRAS mutant and PTEN wild-type cells in NSCLCs.
  • DHEA is more sensitive to TP53 wild-type or G6PD-deficiency mutant in CRC cells.
  • the present invention provides a combination or pharmaceutical composition for treating an anti-cancer drug-resistant cancer with high expression of CES1/2 cells, comprising a therapeutically effective amount of a chemotherapeutic drug and DHEA or its derivatives or metabolites that provides an efficacy in enhancing sensitivity of cancer cells to the chemotherapeutic drug.
  • the NSCLC is KRAS mutant and PTEN wild-type cells.
  • a method for treating a NSCLC or an anti-cancer drug-resistant NSCLC in a subject comprises providing a cancer cell sample from said subject to determine whether the NSCLC is KRAS mutant or PTEN wild-type cells, and administering said subject a therapeutically effective amount of Dehydroepiandrosterone (DHEA) or its derivatives or metabolites, together in combination with a combination with a chemotherapy drug, with a pharmaceutically acceptable carrier, if the cancer cells are KRAS mutant or PTEN wild-type cells.
  • DHEA Dehydroepiandrosterone
  • a method for treating a CRC or an anti-cancer drug-resistant CRC in a subject comprises providing a cancer cell sample from said subject to determine whether the cancer cells are TP53 wild-type or G6PD-deficiency mutant, and administering said subject a therapeutically effective amount of Dehydroepiandrosterone (DHEA) or its derivatives or metabolites, together with a pharmaceutically acceptable carrier, if the cancer cells are TP53 wild-type or G6PD-deficiency mutant.
  • DHEA Dehydroepiandrosterone
  • DHEA have an efficacy in suppressing the growth of cancer stem-like cells through downregulating WNT/ ⁇ -catenin signaling pathway, wherein SRB assay and sphere formation assay were used to examine cellular viability and cancer stem cell-like phenotype, respectively.
  • SRB assay and sphere formation assay were used to examine cellular viability and cancer stem cell-like phenotype, respectively.
  • the expressions of stemness related factors were measured by RT-qPCR and western blotting. It is indicated in the present invention that DHEA reduced HNSCC cell viability, suppressed sphere formation, and inhibited the expression of cancer-stemness markers, such as BMI-1 and Nestin.
  • DHEA that provides an efficacy in enhancing sensitivity of cancer cells to the chemotherapeutic drug as revealed by reduced cell viability, sphere formation, expression of stemness markers, and activation of the WNT pathway.
  • G Luciferase reporter assay showing transcriptional activity of stemness-related markers, including TCF/LEF (WNT) , Nanog, OCT4, and Notch1 in CAL 27 and SAS cells after treatment with 200 ⁇ M DHEA for 24 h.
  • Data represent mean ⁇ standard deviation (SD) derived from three independent experiments. *p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001, compared to control (1 %DMSO only) using t-test.
  • H&E Hematoxylin and eosin staining results of tumors from mice following vehicle and drug treatment.
  • Figure 9 illustrates that the IC50 of DHEA and its analogs on NSCLC cell lines. NSCLC cells were treated with different kinds of DHEA analogs or DHEA with increasing concentrations and cytotoxicity was determined by sulforhodamine B (SRB) assay.
  • SRB sulforhodamine B
  • Figure 10 illustrates that the DHEA has anti-cancer stem cell activity.
  • CL141 cells and CL97 cells were dissociated and seeded 10,000 cells/well in 24-well ultralow attachment plates in DMEM/F12 medium content 1%N2 supplement, EGF (20 ng/mL) and bFGF (20 ng/mL) and cultured with indicated DHEA and its analogs for 7 days and sphere numbers were counted through microscope.
  • Figure 11 illustrates that the Summary of synergistic cytotoxic effects of DHEA and chemotherapeutic agent combinations against lung or ovarian cancer cell lines.
  • Various NSCLC cells were seeded into 96 well plates at density of 3x10 4 /mL 24 hours before drug treatment. Increasing drug concentrations were applied for optimal cancer killing condition and cell viability was measured by Alamar blue assay. These data were then applied to calculate the combination index (CI) via Isobologram analysis in CompuSyn software. +: mild synergistic effect; ++: synergistic effect; -: non-synergistic effect.
  • FIG. 12 illustrates that DHEA has tumor-inhibitory effects in lung cancer in vivo.
  • DHEA and Alimta (pemetrexed) was administrated into NOD-SCID mice in a daily basis (5 mg/kg for each compound) , respectively.
  • Tumor size was measured weekly and
  • body weight was also monitored to check if there was any side effect.
  • Figure 13 illustrates that the anti-tumor effects of DHEA on CRC cell lines.
  • A The IC50 of DHEA on CRC cell lines. CRC cells were treated with DHEA with increasing concentrations and cytotoxicity was determined by SRB assay.
  • B TOP/FOP luciferase assay was used to examine DHEA's ability to suppress ⁇ -catenin-TCF/LEF transcriptional activity in CRC cells.
  • Figure 14 illustrates that the anti-CRC effects of DHEA were independent of p53 and G6PD.
  • A The IC50 of DHEA on HCT116 cells was determined with or without p53 knockout (KO) and/or G6PD knockdown (KD) conditions, as indicated in the table. HCT116 cells were treated with increasing concentrations of DHEA, and cytotoxicity was assessed using the SRB assay.
  • B The Western blot data demonstrated that DHEA induced p21 expression through a pathway independent of both p53 and G6PD.
  • Figure 15 illustrates that the efficient reduction of DLD-1 and DLD-1 5-FU-resistant (DLD-1R) cells'cancer stem cell-like (CSC-like) sphere formation by DHEA.
  • DLD-1R DLD-1 5-FU-resistant
  • CSC-like cancer stem cell-like
  • Figure 16 illustrates that DHEA treatment could mitigate cachectic conditions in mice bearing CT-26 tumors.
  • the co-administration of DHEA appeared to partly enhance the improvement of (A) body weight and (B) grip strength in mice undergoing FOLFOX treatment.
  • the present invention indicates that DHEA exerts anticancer effects, especially regarding the inhibitory effect of cancer stem-like cells, via downregulation of the WNT pathway in vitro and reduces tumorigenicity in vivo. Furthermore, DHEA enhances the therapeutic efficacy of a chemotherapeutic drug, such as IRN, against cancer.
  • a chemotherapeutic drug such as IRN
  • the combination treatment showed increased tumor growth inhibition in both subcutaneous and orthotopic mouse models.
  • HNSCCs Head and neck squamous cell carcinomas
  • HNSCCs Head and neck squamous cell carcinomas
  • HNSCC is the sixth most common cancer worldwide. Approximately 650,000 new cases of HNSCC are diagnosed every year, and it accounts for about 5%of all cancer-related deaths [1, 2] .
  • the standard treatment for HNSCC includes surgery, radiotherapy, chemotherapy, and combinations of these modalities.
  • the survival rate of patients with HNSCC remains low because of drug resistance, tumor metastasis, and recurrence [3] . Therefore, it is critical to understand the mechanisms of local recurrence, metastasis, and resistance that may significantly improve the treatment outcomes of patients with HNSCC.
  • NSCLC Non-small cell lung cancer
  • NSCLC Non-Small Cell Lung Cancer
  • SCLC small cell lung cancer
  • NSCLC non-small cell lung cancer
  • SCLC small cell lung cancer
  • NSCLC non-small cell lung cancer
  • the most common types of NSCLC are squamous cell carcinoma, large cell carcinoma, and adenocarcinoma, but there are several other types that occur less frequently, and all types can occur in unusual histological variants.
  • NSCLC is usually less sensitive to chemotherapy and radiation therapy than SCLC. Patients with resectable disease may be cured by surgery or surgery followed by chemotherapy. Despite advancements in diagnosis and treatment, the overall 5-year survival rate for lung cancer remains poor, with less than 15%survival rate. Conventional therapies like chemotherapy and radiotherapy often yield unsatisfactory outcomes in lung cancer patients, and drug resistance represents a significant unmet clinical need.
  • CRC Colorectal cancer
  • CRC Colorectal cancer
  • bowel cancer also known as bowel cancer, colon cancer, or rectal cancer
  • CRC is the development of cancer from the colon or rectum (parts of the large intestine) .
  • CRC ranks as the fourth most commonly diagnosed cancer worldwide and is a leading cause of cancer-related mortality.
  • Standard treatment for advanced CRC involves chemotherapy regimens based on 5-fluorouracil (5-FU) and oxaliplatin.
  • 5-fluorouracil 5-fluorouracil
  • oxaliplatin oxaliplatin
  • the persistence of chemotherapy resistance poses a significant challenge in CRC management, as reflected by the low 5-year survival rate of only 12%for stage IV disease.
  • CSCs Cancer stem cells
  • Cancer stem cells are a subpopulation of cancer cells that possess self-renewal capacity and pluripotency. CSCs are involved in tumor development, cell proliferation, and metastasis, and are the key “seeds” for tumor initiation, metastasis, and resistance to chemo-and radiotherapies [1, 3-5] . These processes are regulated by several key transcription factors involved in cancer stemness and sphere formation, such as OCT4, Nanog, SOX2, KLF4, and MYC. Additionally, many signaling pathways, such as the WNT and Notch pathways, also contribute to the development of cancer stemness [6-10] .
  • the WNT signaling pathway is involved in cell proliferation, survival, and progression, and influences the self-renewal of stem cells under physiological and pathological conditions [11, 12] .
  • unphosphorylated ⁇ -catenin translocates into the nucleus and subsequently triggers TCF/LEF-mediated transcription of downstream genes, such as CCND1, MYC, and CD44.
  • Dysregulation of the WNT/ ⁇ -catenin signaling pathway is strongly associated with tumorigenesis and progression by maintaining cancer stemness [13] .
  • Recent studies have focused on the therapeutic potential of agents targeting WNT signaling for cancer treatment in mono-or combination therapy [14] .
  • Irinotecan is a topoisomerase I inhibitor that has anticancer activity in solid tumors, such as metastatic colorectal and lung cancer. IRN showed some clinical benefit in recurrent or metastatic HNSCC (R/M HNSCC) [15-17] . IRN is a prodrug that is converted into the active metabolite SN-38 by carboxylesterase (CES) 1 or 2. CES1 was found to be a poor prognostic marker for HNSCC in TCGA HNSCC cohort [18] . It was upregulated in patients with poor prognosis and represented a good therapeutic target for IRN therapy. IRN mono-and combination therapies with other chemotherapeutic agents have been shown to improve the treatment response in cancer patients.
  • CES carboxylesterase
  • IRN In patients with R/M HNSCC, IRN showed a modest overall response rate of 21.2%and a 1-year survival rate of 30.2%. Response to IRN and its toxic side effects appeared to be dose-dependent [19] . Furthermore, the combination of IRN with cisplatin showed synergistic anticancer effect in a phase II trial [20] and the cisplatin/tegafur/uracil/irinotecan triple combination therapy demonstrated a moderate response in patients with R/M HNSCC [16] . Toxicity to patients was tolerable, and the quality of life of the patients improved [16] . However, IRN also induces side effects, such as diarrhea and neutropenia, which can be resolved by optimizing the treatment dosage or increasing the target specificity.
  • DHEA Dehydroepiandrosterone
  • DHEA Dehydroepiandrosterone
  • DHEA-S DHEA sulfate
  • DHEA and DHEA-S are both the most abundant steroids in the human serum at young age and are precursors of sex hormones, such as estrogen and androgen.
  • DHEA has been used as a dietary supplement and reported to have several beneficial effects such as anti-obesity, hypoglycemia, anti-atherosclerosis, anti-aging, and memory-enhancing effects [23-25] .
  • DHEA had an inhibitory effect on HNSCC viability and was less toxic to normal cells, HOF (see Figures 1A and 1B) . Accordingly, DHEA has a potential to develop an anti-cancer drug as having the effect on HNSCC and cancer stemness.
  • DHEA decreased the spheroid size of breast cancer.
  • the effect of DHEA on cancer stemness-related events and the underlying mechanisms have never been studied and is first disclosed in the present invention.
  • DHEA suppressed cancer stemness properties of HNSCC, including decreased sphere size and transcriptional activities of stemness-related transcription factors, such as WNT (TCF/LEF) , Nanog, and OCT4.
  • WNT TNF/LEF
  • Nanog RNA/LEF
  • OCT4 and Nanog are pluripotent transcriptional factors that contribute to maintenance of stemness and cancer progression [49, 50] .
  • the expression of OCT4 and Nanog was slightly decreased following the DHEA treatment, their transcriptional activities were significantly decreased by DHEA, indicating that DHEA has the ability to inhibit CSC potential (see Figures 1C-1G) .
  • DHEA inhibited cancer stem cell-like traits via downregulation of the WNT pathway in HNSCC.
  • DHEA was used in a combination therapy in the present invention. It was found that mRNA levels of CES1/2, encoding the enzymes involved in generation of the active form of IRN, were higher in CAL 27 spheres than in their parental cells ( Figure 3A) , which suggests that the spheres may be more sensitive to IRN than parental cells due to their higher CESs.
  • the human HNSCC cell line, CAL 27, was obtained from the American Type Culture Collection (ATCC, USA) , and HSC-3 and SAS were obtained from the Japanese Collection of Research Bioresources Cell Bank (JCRB, Japan) .
  • the human oral fibroblasts (HOF) were obtained from the ScienCell Research (USA) .
  • the lentivirus packaging cell line human embryonic kidney (HEK) -293T was also obtained from the ATCC.
  • Trans-dehydroepiandrosterone (Sigma #D4000) was dissolved in dimethyl sulfoxide (DMSO) and maintained in 1%DMSO in the medium during in vitro drug treatment at 0-400 ⁇ M.
  • Irinotecan (IRN) used for in vitro studies was purchased from Sigma (#I1406) and treated with cells from 0-10 ⁇ M.
  • (irinotecan hydrochloride trihydrate) used for animal administration was obtained from Pfizer.
  • IP intraperitoneal
  • Cells were plated at 2000 cells/well in a 96-well microplate. Following drug treatments for the desired periods, cells were fixed with 10%trichloroacetic acid (w/v) for 1 h at 4°C, washed with water, and air-dried. SRB solution (0.4% [w/v] in 1%acetic acid) was used to stain the cells for 1 h and then 1%acetic acid was used to wash and remove the excess dye. After adding 20 mM Tris-base, the optical density (OD) of the protein-bound dye was measured at 540 nm to obtain the absorbance. Cell viability was normalized to the control, and the IC50 was calculated using GraphPad Prism 7 software.
  • the synergistic effect assessment was performed by CompuSyn software (https: //www. combosyn. com/) according to the user instruction.
  • Nuclear and cytosolic extracts were isolated from cells using the rapid, efficient and practical (REAP) method [41] . Briefly, following drug treatment, cells were scraped with cold phosphate buffered saline (PBS) and suspended in ice-cold 0.1%NP-40. After pipetting and centrifugation, half of the supernatant was transferred to a new tube and diluted with 4X SDS sample buffer, which was the cytoplasmic fraction. The remaining cell pellet was washed twice with ice-cold 0.1%NP-40 and resuspended with 1X SDS sample buffer diluted in 0.1%NP-40, which constituted the nuclear fraction.
  • RRP rapid, efficient and practical
  • cytoplasmic fraction and the nuclear fraction from each treatment were conducted western blotting assay as described in 2.6 section.
  • a-tubulin was used as a cytoplasmic control; lamin A/C was used as a nuclear fraction control.
  • the expression signals were visualized using the Immobilon Western Chemiluminescent HRP Substrate (Millipore #WBKLS0500) and detected using the Fujifilm LAS4000 luminescent image analysis system. Protein levels were quantified using ImageJ, and the expression was normalized to that of the internal control ( ⁇ -actin) .
  • the antibodies used in this study are listed in Table 2.
  • the target cells were selected in puromycin (1 mg/mL, Invitrogen) for 48 h.
  • puromycin (1 mg/mL, Invitrogen) for 48 h.
  • the stable cells were treated with the drugs, and then promoter activity was measured using ONE-Glo Luciferase Assay System (Promega) .
  • the total RNA (2 mg) was used as a template for reverse transcription performed with a SuperScript III kit (Invitrogen) .
  • the cDNA was subjected to RT-qPCR in triplicate using Omics Green qPCR Master Mix and Gunster MB-P08A 8-strip PCR tubes (Gunster Biotech Inc., Taiwan) .
  • the primers used are listed in Table 3.
  • the relative expression was obtained using the comparative Ct method after normalization to the expression of GAPDH in the StepOne TM Real-Time PCR System.
  • H&E Hematoxylin and eosin
  • IHC immunohistochemical
  • Tumor sections were formalin-fixed and paraffin embedded. H&E or IHC staining was performed using a Discovery XT automated immunostainer (Ventana Medical System) . After dewaxing, deparaffinization, and rehydration, Tris-EDTA buffer was used for antigen retrieval. The sections were immunostained for PCNA (GTX #100539, 1: 500, GeneTex, USA) and Ki67 (Dako #M7240, 1: 150, DAKO/Agilent, Santa Clara, CA) , and subsequently counterstained with hematoxylin.
  • PCNA PCNA
  • Ki67 Dako #M7240, 1: 150, DAKO/Agilent, Santa Clara, CA
  • HNSCC cell lines including CAL 27, SAS, and HSC-3 were treated with different doses of DHEA for 24, 48, and 72 h, respectively.
  • DHEA significantly inhibited cell viability in a time and dose-dependent manner (Figure 1A) .
  • the half maximal inhibitory concentration (IC50) of DHEA was found to be 192.2 ⁇ 28.4 ⁇ M for CAL 27 cells, 292.9 ⁇ 43.9 ⁇ M for SAS cells, and 211.5 ⁇ 13.5 ⁇ M for HSC-3 cells at 72 h.
  • HEF normal human oral fibroblast
  • DHEA also decreased stemness-related mRNA levels, including ALDH1A3, BMI-1, KLF4, and SOX2, after 6 h of treatment in CAL 27 and SAS parental cells (Figure 1D) as well as in spheroid cells ( Figure 1E) .
  • DHEA treatment resulted in a slight reduction in the protein expression of BMI-1 and Nestin but did not affect OCT4 and Nanog expression ( Figure 1F) .
  • WNT TNF/LEF
  • IRN a topoisomerase I inhibitor
  • IRN is a chemotherapeutic drug currently used for the treatment of colorectal cancer [46] .
  • IRN has been used in mono-and combination therapy along with other chemotherapeutic agents in patients with HNSCC and has shown improvement in patient response [18, 20] .
  • IRN is converted to its active form, SN-38, by CES1/2 enzymes in patients [47] .
  • CES1/2 enzymes in patients [47] .
  • Recent studies have shown that the activity and expression of CES are related to IRN efficacy in lung cancer cell lines [17, 48] and solid tumors [49–51] .
  • the combination significantly decreased CAL 27 sphere size compared to DHEA or IRN alone ( Figure 3D) .
  • the combination treatment showed a greater inhibitory effect on the expression of stemness markers, including BMI-1, OCT4, and Nanog ( Figure 3E) .
  • the combination treatment further decreased the expression of active non-phosphorylated ⁇ -catenin and downstream targets, such as CCND1 and CD44 from the whole cell lysate ( Figure 3F) .
  • CAL 27 cells were subcutaneously injected into the flank of immunodeficient mice to establish xenograft models.
  • DHEA (10 mg/kg/twice a week) and/or IRN (50 mg/kg/once a week) were administered via intraperitoneal injection (Figure 4A) .
  • the combination treatment showed greater inhibitory effect on tumor size and weight compared to the controls ( Figures 4B, 4C, 4E) .
  • the combination treatment alleviated irinotecan-induced loss of body weight, suggesting that DHEA may reduce the side effects of IRN ( Figure 4D) .
  • NSCLC cell lines A549, A549-ON, CL141, H441, CL152, HCC827, CL97, and H1975.
  • DHEA metabolites structurally similar to DHEA with slight modifications in one or two functional groups, to assess their toxicities on these NSCLC cells ( Figure 9) .
  • the human lung cancer cell line NCI-H441 obtained from ATCC was subcutaneously injected into the right flank of NOD/SCID mice (4-6 weeks of age) at a concentration of 1 million cells.
  • the test drugs, DHEA and Alimta were administered at a dosage of 5 mg/kg via intraperitoneal injection, five days per week.
  • the present invention indicates that DHEA exerts anticancer effects, especially with regard to the inhibitory effect of cancer stem-like cells, via downregulation of the WNT pathway in vitro and reduces tumorigenicity in vivo. Furthermore, DHEA enhances the therapeutic efficacy of IRN against HNSCC cells. The combination treatment showed increased tumor growth inhibition in both subcutaneous and orthotopic mouse models. These results highlight the synergistic effects of DHEA and IRN. Our results provide a novel and promising therapeutic strategy for patients with HNSCC, NSCLC and CRC. In addition, treatment of DHEA is more sensitive to high expression of CES1/2 cells when DHEA used alone or in combination with IRN.
  • DHEA Treatment of DHEA is more sensitive to KRAS mutant and PTEN wild-type cells in combination with chemotherapy drugs in NSCLC cells. Finally, TP53 wild-type or G6PD deficiency (mutation or down-regulation) is more sensitive to DHEA treatment in CRC cells.

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Abstract

La présente invention concerne une méthode de traitement d'un cancer ou d'un cancer résistant aux médicaments anticancéreux chez un sujet par la suppression de la croissance des cellules de type cellules souches cancéreuses et la régulation à la baisse de la voie WNT. L'invention concerne également une combinaison ou une composition pharmaceutique destinée au traitement d'un cancer ou d'un cancer résistant aux médicaments anticancéreux.
PCT/CN2023/102178 2022-06-24 2023-06-25 Procédé de traitement d'un cancer par suppression de la croissance de cellules souches cancéreuses, et régulation à la baisse de la voie wnt Ceased WO2023246940A1 (fr)

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US20130064815A1 (en) * 2011-09-12 2013-03-14 The Trustees Of Princeton University Inducing apoptosis in quiescent cells
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Title
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