Human Apoptosis Antibody Array Research Articles

Anticancer Activity and Molecular Mechanisms of Acetylated and Methylated Quercetin in Human Breast Cancer Cells

Quercetin, a flavonoid polyphenol found in many plants, has garnered significant attention due to its potential cancer chemoprevention. Our previous studies have shown that acetyl modification of the hydroxyl group of quercetin altered its antitumor effects in HepG2 cells. However, the antitumor effect in other cancer cells with different gene mutants remains unknown. In this study, we investigated the antitumor effect of quercetin and its methylated derivative 3,3′,4′,7-O-tetramethylquercetin (4Me-Q) and acetylated derivative 3,3′,4′,7-O-tetraacetylquercetin (4Ac-Q) on two human breast cancer cells, MCF-7 (wt-p53, caspase-3-ve) and MDA-MB-231 (mt-p53, caspase-3+ve). The results demonstrated that 4Ac-Q exhibited significant cell proliferation inhibition and apoptosis induction in both MCF-7 and MDA-MB-231 cells. Conversely, methylation of quercetin was found to lose the activity. The human apoptosis antibody array revealed that 4Ac-Q might induce apoptosis in MCF-7 cells via a p53-dependent pathway, while in MDA-MB-231 cells, it was induced via a caspase-3-dependent pathway. Furthermore, an evaluation using a superoxide inhibitor, MnTBAP, revealed 4Ac-Q-induced apoptosis in MCF-7 cells in a superoxide-independent manner. These findings provide valuable insights into the potential of acetylated quercetin as a new approach in cancer chemoprevention and offer new avenues for health product development.

Abstract 7308: Induction of apoptosis via the regulation of pro-apoptotic and anti-apoptotic proteins in pancreatic adenocarcinoma BxPC-3 cells by Chinese medicinal herbs Scutellaria barbata and Oldenlandia diffusa

Abstract Apoptosis is a controlled program cell death process that the body uses to combat cancer development. Pancreatic cancer is a devastating disease with a poor prognosis. It is usually symptom-free during development and is advanced at diagnosis. BxPC-3 is a pancreatic adenocarcinoma cell derived from a 61-year-old female with adenocarcinoma. Scutellaria barbata (SB) and Oldenlanda diffusa (OD) have been used in traditional Chinese medicine for treating liver, lung, and rectal cancers. They are included in most herbal cancer treatment formulas in Taiwan hospitals. We previously demonstrated that aqueous extracts of SB and OD selectively induced apoptosis in colon HCT 116 cancer cells but not in colon CCD 841 CoN normal epithelial cells by modulating the pro-apoptotic and anti-apoptotic proteins. They also induced and regulated apoptosis in pancreatic ductal carcinoma PANC-1 Cells. This study used the green/red/blue fluorescent Apoptosis/Necrosis Detection Kit and the Human Apoptosis Antibody Array - Membrane [43 Targets] Test (Abcam). Our fluorescent apoptosis data showed that a 2-hour treatment with 1.5 mg/mL and 3.0 mg/mL aqueous extract of SB and OD induced a statistically significant percentage of apoptosis in BxPC-3 cells (SB: 42.5 ± 3.5%, 48.5 ± 0.5% > 14.0% ± 4.0%, p < 0.05; OD: 72.0 ± 2.0%, 37.5 ± 1.5%> 14.0% ± 4.0%, p < 0.05) as compared to the negative control respectively. Similar results were obtained with the 4-hour incubation period (SB: 46.0 ± 8.0%, 54.5 ± 2.5% > 9.5% ± 2.5%, p < 0.05; OD: 49.0 ± 7.0%, 37.0 ± 0.0%> 9.5% ± 2.5%, p < 0.05). From the antibody array-membrane experiments, significant up-regulation of various pro-apoptotic proteins such as Bid, Bim, Bad, Bax, C3, C8, CD40, HTRA, IGFBP-4, IGFBP-4, p53, TNF-α, and TRAIL-2 as compared to the negative controls were observed; and regulation of anti-apoptotic proteins HSP27, IGF-II, and Survivin were also revealed. These results suggest that SB and OD contain phytochemicals that induce apoptosis in pancreatic adenocarcinoma BxPC-3 cells by regulating various apoptotic protein productions. Further study of their specific modulation effects of apoptosis is warranted to reveal their potential chemopreventive and therapeutic properties against pancreatic and other cancers. Citation Format: Christine Choi, Sarah Wolf, Rekha Isaac, Elim Choi, Abigail Shim, Angel Campana, Padma T. Uppala, Ryan Hayes, Brian Yuen Yau Wong. Induction of apoptosis via the regulation of pro-apoptotic and anti-apoptotic proteins in pancreatic adenocarcinoma BxPC-3 cells by Chinese medicinal herbs Scutellaria barbata and Oldenlandia diffusa [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7308.

POLE2 Regulates Apoptosis of Oral Squamous Cell Carcinoma Cells through the PI3K/AKT Signaling Pathway.

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of the head and neck, but its occurrence and progression mechanisms remain unclear. In addition-there is a lack of effective targeting drugs. The second major subunit of DNA polymerase (POLE2) catalyzes the prolongation of new strand replication and modifies exonuclease domain activity. Our previous study found that POLE2 was associated with OSCC progression, but the mechanism remains unclear. The expression of POLE2 in OSCC tissues was detected using immunological assays. Mann-Whitney U analysis was used to investigate the relationship between POLE2 gene expression and tumor classification and prognosis of OSCC. POLE2 expression was inhibited in OSCC cells, and the effects of gene and protein expression were detected using RT-PCR and Western blotting. The POLE2 knockout model was constructed by transfecting a lentiviral vector. Cell proliferation, apoptosis, and migration were detected using various assays including colony formation, MTT, flow cytometry, wound healing assay, Transwell assay, and the Human Apoptosis Antibody Array. The animal model of OSCC was established by subcutaneous injection of transfected HN6 into 4-week-old female nude mice. After 30 days, tumors were removed under anesthesia and tumor weight and dimension were recorded. Tumor cell proliferation was analyzed using Ki67 staining. POLE2 gene levels were significantly higher in the OSCC tissues than in the normal tissues. In addition, POLE2 gene levels were statistically correlated with tumor classification and prognosis. Silencing POLE2 inhibited the proliferation of oral cancer cells and promoted apoptosis in vitro. Animal experiments also supported a positive correlation between POLE2 and OSCC tumor formation. We further demonstrated that POLE2 could upregulate the expression of apoptosis-related proteins such as caspase-3, CD40, CD40L, DR6, Fas, IGFBP-6, p21, and SMAC. In addition, POLE2 regulated OSCC development by inhibiting the PI3K/AKT signaling pathway. POLE2 is closely related to the progression of OSCC. Thus, POLE2 may be a potential target for OSCC treatment.

Knockdown of KIF15 suppresses proliferation of prostate cancer cells and induces apoptosis through PI3K/Akt signaling pathway

Prostate cancer is one of the most common malignancies in men, which has been considered a public health threat. KIF15 is a kind of driver protein, and its abnormal expression is closely related to the occurrence and development of malignant tumors. The purpose of the study was to explore the significance and role of KIF15 in prostate cancer and to show some potential value for prostate cancer. Immunohistochemistry analysis showed that KIF15 was highly expressed in prostate cancer tissues, which was also positively correlated with T Infiltrate. The loss-of-function and gain-of-function assays based on prostate cancer cells indicated that the change in KIF15 expression could significantly affect cell proliferation, tumorigenesis, migration, and cell apoptosis. The inhibition of prostate cancer development by KIF15 knockdown was also assured in vivo. The Human Apoptosis Antibody Array showed that CD40L, cytoC, DR6, and p21 were up-regulated upon KIF15 knockdown, while IGF-I and Survivin were down-regulated. Moreover, the involvement of the PI3K/Akt pathway in the KIF15-mediated regulation of prostate cancer was preliminarily proved. In summary, KIF15 was identified to play an important role in the development or biological progress of prostate cancer and is considered to possess the potential to be used as a therapeutic target.

Abstract 2540: Modulation of apoptosis in pancreatic adenocarcinoma BxPC-3 cells and ductal carcinoma PANC-1 cells by aqueous extract of Bryophyllum pinnatum

Abstract Bryophyllum pinnatum (BP) has been widely used in tropical regions as traditional medicine. It is reported to have antioxidant, immunomodulatory, antidiabetic, anti-inflammatory, antihypertensive, wound healing, cytotoxic, and antitumor-promoting activities. Its major secondary metabolites include bufadienolides, flavonoids, triterpenes, and steroids. Pancreatic cancer is usually symptom-free during development and is advanced by the time of diagnosis with a poor prognosis. BxPC-3 is a pancreatic adenocarcinoma cell derived from a 61-year-old female with adenocarcinoma while PANC-1 is a pancreatic ductal carcinoma cell derived from a 56-year-old male with adenocarcinoma in the head of the pancreas and metastasized to the duodenal wall. Apoptosis is a controlled program cell death process that the body uses to combat cancer development. We previously showed that an aqueous extract of BP effectively modulated apoptosis in glioblastoma U-87 MG brain cancer cells. It also induced apoptosis in colon HCT 116 cancer cells but not in colon CCD 841 CoN normal epithelial cells by modulating the pro-apoptotic and anti-apoptotic proteins. In this study, we used the green/red/blue fluorescent Apoptosis/Necrosis Detection Kit and the Human Apoptosis Antibody Array - Membrane [43 Targets] Test (Abcam) to assess the effect of BP in the induction and modulation of apoptosis on BxPC-3 and PANC-1 cells. Our fluorescent apoptosis data showed that a 2-hour treatment with 1.5 mg and 3.0 mg aqueous extract of BP induced a statistically significant percentage of apoptosis in BxPC-3 and PANC-1 cells (74.0 ± 1.0%, 90.0 ± 1.0% > 14.0% ± 4.0%, p < 0.05; 85.0 ± 3.0%, 96.5 ± 0.5% > 11.5%± 3.5%, p < 0.05) as compared to the negative control respectively. Similar results were obtained with the 4-hour incubation period (57.0 ± 1.0%, 78.5 ± 6.5% > 9.5% ± 2.5%, p < 0.05; 89.0 ± 1.0%, 85.5 ± 5.5% > 18.2% ± 2.0%, p < 0.05). From the antibody array-membrane experiments, modulation of various apoptosis markers by BP such as the pro-apoptotic proteins Bad, Bax, p27, and p53 as compared to the negative controls were revealed; and regulation of anti-apoptotic proteins Bcl-2, IGF-I, and p21 were also observed. These results suggest that BP contains phytochemicals that induce apoptosis in pancreatic adenocarcinoma BxPC-3 and PANC-1 cells via the modulation of various apoptotic protein productions. Further study of their specific modulation effects of apoptosis is warranted to reveal the potential chemopreventive and therapeutic properties of BP against pancreatic cancer. Citation Format: Jasmine Cha, Elim Choi, Jessica Yi, Sarah Wolf, Rekha Isaac, Min S. Kang, Allana Benjamin, Christine Choi, Ryan Hayes, Brian Yuen Yau Wong. Modulation of apoptosis in pancreatic adenocarcinoma BxPC-3 cells and ductal carcinoma PANC-1 cells by aqueous extract of Bryophyllum pinnatum [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2540.

Abstract 6125: Induction and modulation of apoptosis in pancreatic ductal carcinoma PANC-1 cells by Chinese medicinal herbs Scutellaria barbata and Oldenlandia diffusa

Abstract Pancreatic cancer is a devastating disease with a poor prognosis. It is usually symptom-free during development and is advanced by the time of diagnosis. PANC-1 is a pancreatic ductal carcinoma cell derived from a 56-year-old male with adenocarcinoma in the head of the pancreas and metastasized to the duodenal wall. Apoptosis is a controlled program cell death process that the body uses to combat cancer development. Scutellaria barbata (SB) has been used in traditional Chinese medicine for treating liver, lung, and rectal cancers. SB and Oldenlanida diffusa (OD) are included in most of the herbal cancer treatment formulas in Taiwan hospitals. We previously showed that aqueous extracts of SB and OD selectively induced apoptosis in colon HCT 116 cancer cells but not in colon CCD 841 CoN normal epithelial cells by modulating the pro-apoptotic and anti-apoptotic proteins. These two herbs also effectively modulated apoptosis in MDA-MB-157 and 93B breast cancer cells. Green synthesis of gold nanoparticles (AuNPs) of SB demonstrated effective anticancer activity against this PANC-1 cell. This study used the green/red/blue fluorescent Apoptosis/Necrosis Detection Kit and the Human Apoptosis Antibody Array - Membrane [43 Targets] Test (Abcam). Our fluorescent apoptosis data showed that a 2-hour treatment with 1.5 mg and 3.0 mg aqueous extract of SB and OD induced a statistically significant percentage of apoptosis in PANC-1 cells (SB: 57.5 ± 2.5%, 68.0 ± 0.0% > 11.5% ± 3.5%, p < 0.05; OD: 62.0 ± 9.0%, 67.0 ± 11.0%> 11.5% ± 3.5%, p < 0.05) as compared to the negative control respectively. Similar results were obtained with the 4-hour incubation period (SB: 73.5 ± 0.5%, 72.0 ± 2.0% > 18.2% ± 2.0%, p < 0.05; OD: 59.0 ± 1.0%, 73.5 ± 13.5%> 18.2% ± 2.0%, p < 0.05). From the antibody array-membrane experiments, modulation of various apoptosis markers by SB and OD such as the pro-apoptotic proteins Bid, Bim, Bad, Bax, p27, and p53 as compared to the negative controls were observed; and regulation of anti-apoptotic proteins Bcl-2, FasL, IGF-I, and p21 were also revealed. These results suggest that SB and OD contain phytochemicals that induce apoptosis in pancreatic ductal carcinoma PANC-1 cells via the modulation of various apoptotic protein productions. Further study of their specific modulation effects of apoptosis is warranted to reveal their potential chemopreventive and therapeutic properties against pancreatic and other cancers. Citation Format: MinSeo Kang, Allana Benjamin, Christine Choi, Rekha Isaac, Sarah Wolf, Jasmine Cha, Elim Choi, Jessica Yi, Padma T. Uppala, Ryan Hayes, Brian Yuen Yau Wong. Induction and modulation of apoptosis in pancreatic ductal carcinoma PANC-1 cells by Chinese medicinal herbs Scutellaria barbata and Oldenlandia diffusa [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6125.

XTP1 facilitates the growth and development of gastric cancer by activating CDK6.

Hepatitis B virus X protein (XTP1) is overexpressed in tumor tissues and regulates cancer progression. However, the molecular mechanism of XTP1 in gastric cancer (GC) is poorly understood. Hence, we aimed to dissect the underlying role of XTP1 in the development of GC. Lentiviruses were constructed and transfected into GC cells to upregulate or downregulate gene expression. The expressions of proteins in GC cells or tumor tissues were assessed by quantitative reverse transcription polymerase chain reaction (RT-qPCR), Western blotting, immunohistochemistry (IHC) assay, or the Gene Expression Profiling Interactive Analysis (GEPIA) database. Cell proliferation was assessed via methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, Celigo cell counting assay, cell cycle analysis, and colony formation assay. Cell apoptosis was assessed by flow cytometry. The apoptosis-related proteins were evaluated using the human apoptosis antibody array. GC cell migration was detected by scratch wound-healing assays and Transwell migration assays. Potential downstream molecules were identified by the human GeneChip assay combined with bioinformatics analysis. We found that XTP1 is overexpressed in GC tissues and is positively related to its pathological grade. XTP1 knockdown restrained the growth and migration of GC cells, while XTP1 overexpression promoted cell proliferation and suppressed apoptosis. A mechanistic study indicated that XTP1 knockdown inhibited cyclin-dependent kinase 6 (CDK6) expression and that CDK6 might be a potential downstream molecule of XTP1. Further study confirmed that CDK6 depletion also suppressed GC cell proliferation and migration and increased GC cell apoptosis. Moreover, rescue experiments verified that CDK6 knockdown abated the promotion of XTP1 overexpression on GC progression. XTP1 facilitated the development and progression of GC cells by activating CDK6. Therefore, the XTP1-CDK6 axis might be a potential therapeutic target for GC.

DPP3 expression promotes cell proliferation and migration invitro and tumour growth invivo, which is associated with poor prognosis of oesophageal carcinoma.

Dipeptidyl peptidase III (DPP3), a zinc‑dependent metallopeptidase, is upregulated in a variety of malignancies. However, little is known about its roles in the pathogenesis of these malignancies. The present study was designed to investigate the roles of DPP3 in the pathogenesis and progression of oesophageal cancer (EC). The expression level of DPP3 in EC tissues and adjacent normal tissues was detected in 93cases of tissue biopsies collected from patients diagnosed with oesophageal carcinoma by immunohistochemistry. The effect of DPP3 expression on cell proliferation, migration or apoptosis was determined in DPP3‑depleted EC cells created by infection with lentivirus containing short hairpin RNA specific to the human DPP3 mRNA sequence, followed by detection at the cellular level using a Celigo cell count assay, flow cytometry, wound‑healing assay and Transwell assay as well as chip screening with a Human Apoptosis Antibody Array kit, which enables the quantitative detection of 43 apoptosis‑related genes. A xenograft model was applied to detect the tumour growth and invasion of DPP3‑depleted cancer cells in nude mice. The results revealed that DPP3 expression was elevated in EC tissues compared with adjacent non‑tumour tissues, and high DPP3 expression was significantly associated with poor prognosis. DPP3 depletion resulted in reduced cell proliferation and migration and enhanced cell cycle arrest and apoptosis of EC cells and led to the inhibition of tumour growth and invasion in a xenograft model. In addition, DPP3 depletion was associated with the upregulation of the proapoptotic proteins SMAC and p53 and the downregulation of the antiapoptotic proteins clAP‑2, IGFBP‑2 and TRAILR‑4. Finally, DPP3 may promote cell proliferation, migration and survival of EC cells invitro and tumour growth and invasion of oesophageal carcinoma invivo, and thus may serve as a molecular target for tumour therapy.

Effects and mechanisms of GSG2 in esophageal cancer progression.

Esophageal cancer was recognized as one of the malignant tumors with poor prognosis. Germ cell associated 2 (GSG2) has been reported to be of great significance in cell growth and tumor formation. This study aimed to investigate the biological function and molecular mechanism of GSG2 in esophageal cancer. First, relationship between GSG2 expression and tumor characteristics in esophageal cancer patients was analyzed through immunohistochemical (IHC) staining. MTT assay, flow cytometry, cloning formation assay, wound-healing assay and Transwell assay were used to determine proliferation, apoptosis and migration of esophageal cancer cell with GSG2 knockdown in vitro. Expression of apoptosis related proteins and downstream pathway proteins after GSG2 knockdown were detected through Human Apoptosis Antibody Array and western blot analysis. The GSG2 knockdown function in vivo was explored through a xenograft tumor model. GSG2 was highly expressed in tumor tissues, which has clinical significance in predicting the malignant degree of patients with esophageal cancer. In addition, GSG2 knockdown significantly inhibited a variety of malignant biological behaviors of esophageal cancer cells, such as inhibiting proliferation, reducing colony formation, promoting apoptosis, hindering migration. The decrease of GSG2 expression in esophageal cancer cells can inhibit the xenograft tumor growth. In conclusion, GSG2 was involved in esophageal cancer progression and development, which may provide an effective molecular target for the treatment of esophageal cancer in the future.

GSG2 promotes tumor growth through regulating cell proliferation in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is one of the most commonly diagnosed malignant tumors in the world. In recent years, more and more inhibitors against gene targets have been found to be beneficial to survival. However, the function of homo-sapiens histone H3 associated protein kinase (GSG2) in HCC has not been completely understood. MethodsThe expression of GSG2 in HCC tissues was detected by immunohistochemical staining. The lentivirus-mediated short hairpin RNA (shRNA) was used to knockdown GSG2 expression in HCC cell lines Hep3B2.1-7 and SK-HEP-1. Cell proliferation and colony formation were detected by MTT assay and colony formation assay, respectively, and flow cytometry assay was used to investigate the cell apoptosis in vitro. Mice xenograft model was constructed to detect the functions of GSG2 on tumor growth in vivo. Human Apoptosis Antibody Array was conducted to find the possible mechanism. ResultsGSG2 was overexpressed in HCC tissues compared with adjacent normal tissues. The knockdown of GSG2 had the functions of inhibiting the progression of HCC, including inhibiting cell proliferation and colony formation and promoting cell apoptosis. Compared with shCtrl group, the shGSG2 group expressed higher apoptotic genes such as caspase 3, caspase 8, Fas and FasL, while lower IGF1, Bcl2 and Bcl-w. ConclusionsOur study showed that knockdown of GSG2 suppresses the tumor growth in vitro and vivo. Therefore, GSG2 might play an oncogenic role in HCC.

Abstract 6261A: Chinese medicinal herbs Scutellaria barbata and Oldenlandia diffusa modulate apoptosis via induction of pro-apoptotic and anti-apoptotic proteins in MDA-MB-157 and 93B breast cancer cells

Abstract Scutellaria barbata (SB) and Oldenlandia diffusa (OD) have been used in Traditional Chinese Medicine for treating colon cancer, glioblastoma, and ovarian cancer liver, lung, and rectal cancers. These two herbs are included in most of the herbal cancer treatment formulas in China and Taiwan hospitals. We previously demonstrated that aqueous extracts of SB and OD inhibited mutagenesis, DNA binding, and metabolism of aflatoxin B1 and benzo[a]pyrene in carcinogenesis. In studies by Hugo (2006) and Perez (2010), the efficacy of BZL 101 (an extract of SB) has been demonstrated in preliminary Phase Ia and Ib clinical trials in metastatic breast cancer patients. It is also shown to induce an S phase arrest of the cell cycle by affecting the Cycline A2 and CDK2 expression in estrogen-sensitive MCF7 and estrogen insensitive MDA-MB-231 breast cancer cells. MeOH and BuOH extract of OD have been illustrated to reduce MCF7 invasion via the inhibition of MMP-9 and ICAM-1 apoptotic proteins expression. Ursolic acid of OD could inhibit breast cancer metastasis by suppressing glycolytic metabolism through activating SP1/Caveolin-1 signaling. In this study, the effectiveness of aqueous extract of SB and OD in the modulation of apoptosis of breast cancer cell lines MDA-MB-157 and 93B (a drug-resistant APC-mutant of it) were investigated. Assessments were performed using green/red/blue fluorescent Apoptosis/Necrosis Detection Test and the Human Apoptosis Antibody Array - Membrane [43 Targets] Test (Abcam). Our fluorescent apoptosis data showed that 3-hours treatment with 2.5 mg aqueous extract of SB induced a statistically significant percentage of apoptosis in MDA-MB-157 (46.5 ± 7.5% > 16%± 1.0%, p < 0.05) and 93B (46.5 ± 7.5% > 16%± 1.0%, p < 0.05) as compared to the negative control. Similar results were obtained with the OD treatment. From the antibody array-membrane experiments, modulation of various apoptosis markers by SB such as the pro-apoptotic proteins BID and BIM (p=0.019; p=0.007) in MDA-MB-157 and Bad (p=0.002), Bax (p=0.020), BID (p=0.001), p27 (p=0.004), p53 (p=0.022) in 93B as compared to the negative controls; and anti-apoptotic markers BcL-2, FasL, IGF-I, and p21 were also observed. OD treatment also induced similar significant production of these two types of proteins. These results suggest that SB and OD contain phytochemicals that induce apoptosis in MDA-MB-157 and 93B via the modulation of these pro-apoptotic and anti-apoptotic proteins. Citation Format: MinSeo Kang, Nathaly Manrique, Rekha Issac, Sarah Wolf, Jasmine Cha, SungBeen Han, Hugo Jie Qin, Lun Kim, Padma P. Uppala, Brian Yuen Yau Wong. Chinese medicinal herbs Scutellaria barbata and Oldenlandia diffusa modulate apoptosis via induction of pro-apoptotic and anti-apoptotic proteins in MDA-MB-157 and 93B breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6261A.

PSMC2 knockdown inhibits the progression of oral squamous cell carcinoma by promoting apoptosis via PI3K/Akt pathway

ABSTRACT Proteasome 26S subunit, ATPase 2 (PSMC2) is a recently identified gene which is potentially associated with human carcinogenesis. However, the effects of PSMC2 on oral squamous cell carcinoma (OSCC) is still unclear. Here, we investigated PSMC2 expression in OSCC tissues and explored its effects on the biological behaviors of OSCC cells. PSMC2 expression was evaluated by immunohistochemistry in a tissue microarray containing 60 OSCC tissues and 9 normal tissues. PSMC2 was knocked down through lentivirus infection in OSCC cell lines. MTT, colony formation, flow cytometry, transwell, and scratch assays were performed to detect effects of PSMC2 knockdown on phenotypes of OSCC cells. Human apoptosis antibody array was used to screen potential downstream of PSMC2 in OSCC. Finally, the effects of PSMC2 knockdown on tumor growth were assessed in a tumor xenograft model using BALB/c nude mice. PSMC2 expression was significantly upregulated in OSCC tissues compared with normal tissues and correlated with poor prognosis. PSMC2 knockdown significantly suppressed cell proliferation, migration, but promoted apoptosis of OSCC cells. Additionally, we confirmed that PSMC2 knockdown can increase the expression of pro-apoptotic proteins. Furthermore, we found that PSMC2 knockdown downregulated expression of p100, p-Akt, CDK6, and upregulated of MAPK9. Xenograft experiments revealed that PSMC2 knockdown can suppress OSCC tumor growth and promote apoptosis. This study demonstrated that PSMC2 plays a critical role in OSCC progression through affecting pro-apoptotic protein expression and apoptosis pathways. It indicated that targeting PSMC2 might be a promising strategy for OSCC treatment.

Abstracts

Abstracts

Abstract 1923: Modulation of apoptosis in glioblastoma U87-MG cancer cells by aqueous extract of Bryophyllum pinnatum

Abstract Bryophyllum pinnatum (BP) has been used in anthroposophic medicine to treat “hyperactivity diseases” such as tocolytic symptom and overactive bladder syndrome since the beginning of 20th century with no side effect. Its major secondary metabolites include bufadienolides, flavonoids, triterpenes, and steroids. BP has been widely used in tropical regions as traditional medicine. It is reported to have antioxidant, immunomodulatory, antidiabetic, anti-inflammatory, antihypertensive, wound healing, cytotoxic, and antitumor promoting activities. Glioblastoma is an aggressive and lethal tumor of the brain with few treatment options. In this study, the effects of aqueous extracts of BP on the induction and modulation of apoptosis in glioblastoma U87-MG cells were assessed using the green/red/blue fluorescent Apoptosis/Necrosis Detection Kit and the Human Apoptosis Antibody Array - Membrane (43 Targets) test by the Abcam cooperation. Our data revealed that 3-hours of 1 mg/mL and 2 mg/mL BP treatment induced statistically similar amounts of apoptosis observed as green apoptotic cells and red necrotic cells in U87-MG cells respectively (apoptotic cells = 86.0 ± 6%, necrotic cells = 14.0 ± 6.0% ; apoptotic cells = 85.0 ± 4.0% , necrotic cells = 14.5 ± 3.5%). The percent of apoptosis was not significantly different as compared to that of the positive control induced by 1µMol Staurosporine (94.5 ± 0.5%, 5.5 ± 0.5%); while significantly different from the negative control which had none apoptotic cells. Slightly less induction of apoptosis was obtained from 6-hours of 1 mg/mL and 2 mg/mL BP treatment with more necrotic cells (81.5 ± 2.5%, 18.5 ± 2.5%; 78.0 ± 3.0%, 21.5 ± 2.5% respectively). Modulation of various apoptosis markers such as up-regulation of Bax, p53, Caspase 3; and down-regulation of Bcl-2 and Bcl-w was also observed. These results suggest that BP contains phytochemicals which induce apoptosis in glioblastoma U87-MG cells by modulating these apoptotic pathway markers. Further study of the specific modulation effects of BP on apoptosis is warranted to reveal its potential chemopreventive and therapeutic properties against glioblastoma and other cancers. Citation Format: Kristin Ferrer, Joshua Li, Camille Kordas, Jasmine Cha, Sung Been Han, Joon Seo, Nathaly Manrique, Min Seo Kang, Yi Shan Jin, Si Won Choi, Ben Hiramoto, Denise Smith, Brian Yuen Wong. Modulation of apoptosis in glioblastoma U87-MG cancer cells by aqueous extract of Bryophyllum pinnatum [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1923.

Abstract 1926: Selective induction of apoptosis by aqueous extract of Chinese medicinal herbs Scutellaria barbata and Oldenlandia diffusain HCT 116 colon cancer cells and CCD 841 CoN colon epithelial cells

Abstract Scutellaria barbata (SB) and Oldenlandia diffusa (OD) have been used in traditional Chinese medicine for treating liver, lung and rectal tumors. These two herbs are included in most of the herbal cancer treatment formulas in Taiwan hospitals. We previously showed that aqueous extracts of SB and OD inhibited mutagenesis, DNA binding and metabolism of aflatoxin B1 and benzo(a)pyrene. They were also shown to inhibit foci formation in the colon of AOM-induced mice. Other researchers demonstrated their effects in regulation of various apoptotic enzymes and pathways. Ethanol extract of SB was shown to inhibit colorectal cancer growth by suppression of the Wnt/β-catenin signaling pathway while ethanol extract of OD was indicated in the inhibition of proliferation and induction of apoptosis of 5-FU resistant colorectal cancer cells via the regulation of PI3K/AKT signaling pathway. In this study, the effects of aqueous extracts of SB and OD on the induction and modulation of apoptosis in HCT 116 colon cancer cells and CCD 841 CoN normal color epithelial cells were assessed using TUNEL assay, Apoptosis/Necrosis Detection Kit, and Human Apoptosis Antibody Array - Membrane (43 Targets) test (Abcam). Our data demonstrated that both the 3-hours and 6 hours of 1.5 mg/mL SB treatments induced apoptosis in HCT 116 colon cancer cells significantly (77.5 ± 1.5%, 87.5± 3.5%) as compared to the negative control (13.5 ± 0.5%, 17.0 ± 6.0%), p < 0.05. The percent of apoptosis was not significantly different as compared to that of the positive control induced by 1µMol Staurosporine (99.5 ± 0.5%, 91.9 ± 9.0%); while there was no notable induction of apoptosis in normal CCD 841 CoN colon epithelial cells (18.5 ± 1.5%, 14.4 ± 4.0% separately). The majority of the normal cells remained non-induced (71.0 ± 1.0%, 68.5 ± 6.5%). A similar selective induction of apoptosis, in the two cell lines separately, was obtained from the 3-hours and 6 hours of 1.5 mg/mL OD treatments (HCT116 - 44.0 ± 4.0%, 83.5± 15.5%; CCD841 CoN - 6.5 ± 5.5%, 7.0 ± 2.0% respectively). Modulation of various apoptosis markers production such as up-regulation of Bax, BID, Bad, p27, p53, Caspases 8 and 3; and down-regulation of Bcl-2 and Bcl-w was also observed. These results suggested that SB and OD contain phytochemicals which selectively induce apoptosis in HCT 116 cancer cells by modulating these markers while not significantly affecting the normal CCD 841 CoN colon epithelial cells. Further study of their specific modulation effects and phytochemicals on apoptosis is warranted to reveal their potential chemopreventive and therapeutic properties against colon cancer and other cancers. Citation Format: Camille Kordas, Joshua Li, Kristin Ferrer, Yi Shan Jin, Nathaly Manrique, Min Seo Kang, Joon Seo, Sung Been Han, Jasmine Cha, Anthony Miller, Padma Tadi Uppala, Brian Yuen Wong. Selective induction of apoptosis by aqueous extract of Chinese medicinal herbs Scutellaria barbata and Oldenlandia diffusain HCT 116 colon cancer cells and CCD 841 CoN colon epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1926.

CDC42EP3 is a key promoter involved in the development and progression of gastric cancer.

Gastric cancer (GC) is one of the most prevalent cancers and severely endangers human health. Due to the low rate of diagnosis, most patients with GC are diagnosed as advanced. CDC42 effector protein 3 (CDC42EP3) has been revealed to be involved in several types of human cancers' development and progression. However, the function of CDC42EP3 in GC is not yet clear. CDC42EP3 expression was detected by immunohistochemistry, quantitative real-time PCR and Western blot assay in tumor tissues and cell lines of GC. CDC42EP3 knockdown cell models were constructed by lentivirus transfection. Cell proliferation was evaluated by the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The wound-healing assay and the transwell assay were utilized to assess the cell migration. Also, the cell apoptosis and the cell cycle were evaluated by flow cytometry. Moreover, the mechanism was investigated by Human Apoptosis Antibody Array. The in vivo experiments were conducted to verify the effects of CDC42EP3 knockdown on the tumor growth of GC. The expression level of CDC42EP3 was up-regulated in tumor tissues. High CDC42EP3 expression was positively related to more advanced tumor grade. CDC42EP3 knockdown inhibited cell proliferation and migration, promoted cell apoptosis and suppressed the tumor growth. On the other hand, it was also found that the silencing of CDC42EP3 inhibited HSP27 and IGF-1sR expression as well as promoted Caspase3, p53, TNF-α, TNF-β, TRAILR-1 and TRAILR-2 expression. CDC42EP3 was revealed to work as a tumor promoter in the development and progression of GC, which could be a promising therapeutic target for the therapy of GC.

Knockdown of MCM8 inhibits development and progression of bladder cancer in vitro and in vivo

BackgroundBladder cancer is a frequently diagnosed urinary system tumor, whose mortality remains rising. Minichromosome maintenance eight homologous recombination repair factor (MCM8), a newly discovered MCM family member, has been shown to be required for DNA replication. Unfortunately, little is known concerning the roles of MCM8 in bladder cancer.MethodsThe present study, we aimed at probing into the impacts and detailed mechanisms of MCM8 in bladder cancer progression. In this study, MCM8 expression level was detected through immunohistochemistry staining (IHC), qRT-PCR and Western blot assay. Silenced MCM8 cell models were constructed by lentivirus transfection. In vitro, the cell proliferation was evaluated by the MTT assay. The wound-healing assay and the transwell assay were utilized to assess the cell migration. Also, the cell apoptosis and the cell cycle were determined by flow cytometry. Moreover, the Human Apoptosis Antibody Array assay was performed to analyze the alterations of apoptosis-related proteins. The in vivo experiments were conducted to verify the effects of MCM8 knockdown on the tumor growth of bladder cancer.ResultsThe results demonstrated that compared with normal adjacent tissues, MCM8 expression in bladder cancer tissues was strongly up-regulated. The up-regulation of MCM8 expression in bladder cancer may be a valuable independent prognostic indicator. Of note, MCM8 inhibition modulated the malignant phenotypes of bladder cancer cells. In terms of mechanism, it was validated that MCM8 knockdown made Akt, P-Akt, CCND1 and CDK6 levels down-regulated, as well as MAPK9 up-regulated.ConclusionsTaken together, our study demonstrated an important role of MCM8 in bladder cancer and created a rationale for the therapeutic potential of MCM8 inhibition in human bladder cancer therapy.

Eukaryotic Translation Initiation Factor 3 Subunit B Is a Promoter in the Development and Progression of Pancreatic Cancer.

Background: Pancreatic cancer (PC) is a malignant tumor with hidden incidence, high degree of malignancy, rapid disease progression, and poor prognosis. Eukaryotic translation initiation factor 3 subunit B (EIF3B) is necessary for tumor growth, which is an alternative therapeutic target for many cancers. However, little is known about the relationship between EIF3B and PC.Methods: The expression of EIF3B in PC was detected by immunohistochemistry. EIF3B knockdown cell models were constructed by lentivirus infection. The MTT assay, the wound-healing assay, the transwell assay, the flow cytometry, and the Human Apoptosis Antibody Array was used to detect the effects of EIF3B knockdown on cell proliferation, cell migration, cell apoptosis, and cell cycle in vitro. Also, the effects of EIF3B knockdown on the tumor growth of PC were determined in vivo.Results: This study showed that the expression level of EIF3B was significantly up-regulated in PC tumor tissues and associated with pathological grade. In vitro, EIF3B knockdown inhibited the PC cell proliferation and migration, and the apoptosis levels were obviously promoted by regulating apoptosis-related proteins including Bcl-2, HSP27, HSP60, Survivin, sTNF-R2, TNF-α, TNF-β, TRAILR-3, TRAILR-4, and XIAP. Furthermore, the tumor growth of PC was inhibited after the knockdown of EIF3B in vivo.Conclusion: EIF3B was up-regulated in PC and was a promoter in the development and progression of PC, which could be considered as a therapeutic target for the treatment of PC.

UAP1L1 plays an oncogene-like role in glioma through promoting proliferation and inhibiting apoptosis.

Uridine diphosphate-N-acetylglucosamine pyrophosphorylase-1-like-1 (UAP1L1) is involved in protein glycosylation and promotes proliferation in some tumors. By analyzing the publicly available Gene Expression Omnibus (GEO) database, we found that UAP1L1 displayed a significant change between paired glioma and normal brain tissues. The purpose of this study was to investigate the expression and functional role of UAP1L1 in glioma. To determine the expression level of UAP1L1 in glioma, immunohistochemistry (IHC) staining was performed in tissue microarrays of 160 gliomas and 24 normal brain tissues. The correlation between UAP1L1 expression and the outcomes of glioma patients was analyzed. Human glioblastoma cell lines, U251 and U87, were employed in this study. Endogenous UAP1L1 expression in U251 and U87 cells was detected by quantitative real-time polymerase chain reaction (qRT-PCR). A lentiviral short hairpin RNA (shRNA) vector (shUAP1L1) was constructed and used to infect U251 and U87 cells to knock down the expression of UAP1L1. We performed 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay, flow cytometry, human apoptosis antibody array, and in vivo subcutaneous xenograft model to investigate the biological functions of UAP1L1. We revealed that UAP1L1 expression was obviously upregulated in the glioma tissues. The increased UAP1L1 expression level was clinically associated with higher tumor grades and poorer patient prognoses. Moreover, we demonstrated that UAP1L1 knockdown suppressed proliferation and increased apoptosis of glioma cells in vitro. In the xenograft mouse model, we further verified that UAP1L1 knockdown could attenuate the growth of glioma cells in vivo. These results indicated that UAP1L1 may play an oncogene-like role in glioma, especially in high grade glioma, and thus may be of clinical importance as a future therapeutic target.

CCNI2 plays a promoting role in the progression of colorectal cancer.

Colorectal cancer (CRC) is one of the most common malignancies and most of the patients diagnosed with advanced CRC have unsatisfactory treatment effect and poor prognosis. The purpose of this study was to investigate the effect of CCNI2 on the development of CRC. In this sutdy, immunohistochemical staining was used to detect CCNI2 expression levels in clinical samples, meanwhile, the Kaplan‐Meier survival analysis was conducted. Celigo cell counting assay was used for screening shCCNI2s. QPCR and WB were performed to verify knockdown efficiency of CCNI2. Cell proliferation, colony formation, cell cycle, apoptosis, and mechanism investigation of CCNI2 knockdown were investigated by MTT assay, colony formation assay, fluorescence‐activated cell sorting, and human apoptosis antibody array, respectively. Otherwise, the mouse model of CCNI2 knockdown was also constructed. The results of immunohistochemical staining and qPCR indicated that CCNI2 had a high expression level in the CRC tissues and cell lines. Kaplan‐Meier survival analysis manifested that the high expression of CCNI2 suggested poor prognosis. The expression of CCNI2 was significantly reduced by CCNI2‐siRNAs, and the downregulated expression level of CCNI2 inhibited CRC cell proliferation and colony formation, arrested cell cycle in G2 phase, as well as promoted cell apoptosis. The various indexes of solid tumor in mice models indicated that CCNI2 knockdown could suppress the growth of CRC tumor. Based on the comprehensive analysis of the above results, CCNI2 was contributed to the progression of CRC and could serve as a prognostic marker for CRC.