BACKGROUND: The pathogenesis of fibrous epulis is still quite unclear. Our recent genome-wide RNA sequencing analysis revealed that in fibrous epulis, RAS-PI3K-AKT-NF-κB pathway regulates the expression of Bcl-2 family and IAP family genes, leading to increased proliferation and the inhibition of apoptosis. The PI3K/AKT signaling pathway can promote autophagy in human gingival fibroblasts; therefore, the purpose of the present study was to identify whether autophagy is involved in the pathogenesis of fibrous epulis.
METHODS: Differentially expressed genes (DEGs) between fibrous epulis lesions and normal gingival tissues were identified using the PCR array. The expression levels of eighteen autophagy-related (ATG) family genes, twelve B-cell lymphoma 2 (Bcl-2) family genes, and eleven cysteine-dependent aspartate-directed protease (caspase) family genes were validated using quantitative real-time PCR (qRT-PCR). Autophagy induction was determined by measuring microtubule-associated protein light chain 3 (LC3) conversion (LC3-I to LC3-II) by immunoblot analysis.
RESULTS: The PCR array identified six upregulated genes, whereas no genes were expressed at significantly lower levels. The upregulated genes were BCL2, BCL2L1, CXCR4, HSP90AA1, HSPA8, and IGF1, which all belong to the \"regulation of autophagy\" group but not the \"autophagy machinery components\" group. qRT-PCR verified that the expression levels of BCL2, BCL2L1 (also known as BCL-XL), and BCL2L2 (also known as BCL-W) were significantly increased in fibrous epulis. No LC3-I to LC3-II conversion was observed.
CONCLUSIONS: The present study reveals that in fibrous epulis, Bcl-2 and Bcl-xL coordinately mediate gingival cell escape from apoptosis, leading to uncontrolled proliferation. Moreover, ATG family genes are not activated, and autophagy is not involved in this process.

The cysteine-containing aspartate specific proteinase (caspase) family plays important roles in apoptosis and the maintenance of homeostasis in lampreys. We conducted genomic and functional comparisons of six distinct lamprey caspase groups with human counterparts to determine how these expanded molecules evolved to adapt to the changing caspase-mediated signaling pathways. Our results showed that lineage-specific duplication and rearrangement were responsible for expanding lamprey caspases 3 and 7, whereas caspases 1, 6, 8, and 9 maintained a relatively stable genome and protein structure. Lamprey caspase family molecules displayed various expression patterns and were involved in the innate immune response. Caspase 1 and 7 functioned as a pattern recognition receptor with a broad-spectrum of microbial recognition and bactericidal effect. Additionally, caspases 1 and 7 may induce cell apoptosis in a time- and dose-dependent manner; however, apoptosis was inhibited by caspase inhibitors. Thus, these molecules may reflect the original state of the vertebrates caspase family. Our phylogenetic and functional data provide insights into the evolutionary history of caspases and illustrate their functional characteristics in primitive vertebrates.

Osteosarcoma is the most common bone cancer in children and adolescents. Tissue inhibitors of metalloproteinases (TIMPs)-3 inhibit matrix metalloproteinases to limit extracellular matrix degradation. Cisplatin is a widely used chemotherapeutic drug used to cure osteosarcoma. Interleukin (IL)-6 and TIMP3 play important roles in the drug resistance of osteosarcoma; however, their relationship in this process remains unclear. This study aimed to explore the role of TIMP3 in the cisplatin sensitivity of osteosarcoma and its underlying molecular mechanisms in vitro and in vivo. We compared TIMP3 expression levels between patients with cisplatin-sensitive and -insensitive osteosarcoma. TIMP3 was overexpressed or knocked down in the Saos2-lung cell line, which is a Saos2 subtype isolated from pulmonary metastases that has higher cisplatin chemoresistance than Saos2 cells. IL-6 expression, cell proliferation, sensitivity to cisplatin, migration, and invasion after TIMP3 overexpression or knockdown were determined. The same experiments were performed using MG63 and U2OS cells. Subsequently, luciferase-labeled Saos2-lung cells overexpressing TIMP3 were injected into the tibiae of nude mice treated with cisplatin. The results showed that IL-6 inhibited TIMP3 expression in Saos2 and Saos2-lung cells via signal transducer and activator of transcription 3 (STAT3) activation. STAT3 knockdown reversed the effect of IL-6. The expression of TIMP3 was higher in patients with cisplatin-sensitive osteosarcoma than in those with insensitive osteosarcoma. IL-6 expression was downregulated upon TIMP3 overexpression, and upregulated by TIMP3 knockdown. TIMP3 overexpression suppressed cell proliferation and enhanced cisplatin sensitivity by activating apoptosis-related signal pathways and inhibiting IL-6 expression in vitro and in vivo. In conclusion, cisplatin sensitivity correlated positively with TIMP3 expression, which is regulated by the IL-6/TIMP3/caspase pathway. The TIMP3 pathway could represent a target for new therapies to treat osteosarcoma.

Breast cancer is a major life‑threatening malignancy and is the second highest cause of mortality. The aim of the present study was to investigate the effects of tectorigenin (Tec), a Traditional Chinese Medicine, against human breast cancer cells in vitro. MDA‑MB‑231 and MCF‑7 human breast cancer cells were treated with various concentrations of Tec. Cell proliferation was evaluated using the Cell Counting kit‑8 assay, and apoptosis and the cell cycle were examined by flow cytometry. The migratory and invasive abilities of these cells were detected by Transwell and Matrigel assays, respectively. Metastasis‑, apoptosis‑ and survival‑related gene expression levels were measured by reverse transcription‑quantitative polymerase chain reaction and western blotting. The results indicated that Tec was able to inhibit the proliferation of MDA‑MB‑231 and MCF‑7 cells in a dose‑ and time‑dependent manner. Furthermore, Tec treatment induced apoptosis and G0/G1‑phase arrest, and inhibited cell migration and invasion. Tec treatment decreased the expression of matrix metalloproteinase (MMP)‑2, MMP9, BCL‑2, phosphorylated‑AKT and components of the mitogen‑activated protein kinase (MAPK) signaling pathway, and increased the expression of BCL‑2‑associated X, cleaved poly [ADP‑ribose] polymerase and cleaved caspase‑3. In conclusion, Tec treatment suppressed human breast cancer cells through the downregulation of AKT and MAPK signaling and the upregulated expression and/or activity of the caspase family in vitro. Therefore, Tec may be a potential therapeutic drug to treat human breast cancer.

Grape seed proanthocyanidins (GSPs) have been reported to possess a wide array of pharmacological and biochemical properties. Recently, GSPs have been reported to inhibit various types of colorectal cancer; however, the mechanism(s) involved remain unclear. The present study investigated the effects of GSPs on HCT-116 human colorectal carcinoma cell line. Exposure of these cells to GSPs for 48 h resulted in a significant concentration-dependent inhibition of cell viability. Further investigation indicated that GSPs induced apoptosis of these cells. Analyses of mRNA expression levels using reverse transcription-quantitative polymerase chain reaction and protein expression levels by western blotting revealed that this was associated with increased expression levels of p53 tumor suppressor protein, cytochrome c, and pro-apoptotic proteins, apoptosis regulator Bax (Bax) and Bcl-2 homologous antagonist/killer. Furthermore, decreased expression levels of the anti-apoptotic protein, B cell lymphoma-2 and activation of caspase-2, caspase-3 and caspase-9 were demonstrated. GSP-induced loss of mitochondrial membrane potential was also detected by JC-1 assay. These findings suggested that GSPs induced colon cancer cell apoptosis via the mitochondrial signaling pathway. This provided evidence indicating that GSPs may provide potential chemotherapeutic agents for colorectal cancer.

The aim of the present study was to investigate the anticancer activities of Nelumbo nucifera (Ba lotus) stamen ethanol crude extract (BLSEE) in human colon carcinoma HCT-116 cells. MTT assay, flow cytometry analysis and reverse transcription-polymerase chain reaction assay were employed to investigate the anticancer mechanisms of BLSEE (100, 200 and 400 µg/ml) in HCT-116 cells. BLSEE reduced HCT-116 cell proliferation in a dose-dependent manner. BLSEE treatment also significantly increased the sub-G1 population in HCT-116 cells (P=0.0020 at 400 µg/ml), as shown by flow cytometry assay. Following treatment with BLSEE, the mRNA levels of the apoptosis-associated factors Fas, Fas ligand, tumor necrosis factor-related apoptosis-inducing ligand, death receptor 4 (DR4), death receptor 5 (DR5), caspases 3, 8 and 9, and B-cell lymphoma-2 (Bcl-2) associated X protein were increased, and the expression of anti-apoptotic Bcl-2 and Bcl-extra large was decreased in HCT-116 cells. The mRNA levels of matrix metalloproteinase (MMP)-2, MMP-9, TIMP metallopeptidase inhibitor 1 and TIMP metallopeptidase inhibitor 2 were also regulated by BLSEE treatment. In addition, BLSEE was able to modulate the expression of inflammation-associated nuclear factor-κB, inhibitory κBα, inducible nitric oxide synthase and cyclooxygenase 2 in HCT-116 cells. The present study clearly indicated the cytotoxicity of BLSEE in HCT-116 cells through induced cellular apoptosis. These results also suggested the BLSEE may be a powerful agent against colon cancer cells.

To investigate the anticancer activities of alkaloids from the Ba lotus seed (BLSA) in human nasopharyngeal carcinoma (NPC) CNE-1 cells, an MTT assay, flow cytometry, reverse transcription-polymerase chain reaction and western blotting were performed. BLSA was found to significantly reduce CNE-1 cell proliferation in a dose-dependent manner at all concentrations compared with the control (P<0.05). In addition, flow cytometry analysis identified that BLSA treatment significantly increased the sub-G1 content in CNE-1 cells (P<0.05). Following BLSA treatment, the mRNA and protein levels of a number of apoptosis-related factors, such as caspase family members (caspase-3, -8 and -9), B-cell lymphoma (Bcl)-2-associated X protein, Fas and Fas ligand were significantly increased compared with the control (P<0.05). This was accompanied by a significant decrease in anti-apoptotic Bcl-2 and Bcl-extra large protein expression compared with the control (P<0.05). Furthermore, BLSA treatment was determined to modulate CNE-1 cell expression of nuclear factor (NF)-κB and NF-κB inhibitor α. The results of the present study indicate that BLSA has anticancer activity through inducing cellular apoptosis. In addition, these results suggest that BLSA can be used as a therapeutic agent in NPC.

Iso-suillin, an isomer of suillin that belongs to the prenylphenol class of fungal derivatives, was isolated from petroleum ether extracts of Suillus flavus. The IC50 value of iso-suillin in K562 cells was 0.87 μM, which was lower than the positive control cisplatin (19.33 μM). Iso-suillin-treated K562 cells exhibited an increased rate of apoptosis, mitochondrial membrane potential (MMP) depolarization, and G0/G1 arrest. Western blot analysis revealed that these cells displayed significantly upregulated expression of several apoptosis-related proteins, including cytochrome c, caspase 9, FADD (Fas-associating protein with a novel death domain), caspase 8, caspase 3, and Bax. Moreover, the expression of two anti-apoptosis proteins, NF-κB and Bcl-2, was downregulated. Inhibitors of caspase 9 and caspase 8 protected the K562 cells from apoptosis. Taken together, our results suggest that iso-suillin induces K562 apoptosis through the mitochondrial and death receptor pathways and that iso-suillin may represent a candidate anti-leukemia treatment.