α‑Phellandrene (α‑PA), a natural constituent of herbs, inhibits cancer cell viability and proliferation. 5‑Fluorouracil (5‑FU) is a frequently utilized chemotherapeutic medicine for the treatment of colon cancer, which works by triggering cancer cell apoptosis. The present study examined how the combination of α‑PA and 5‑FU affects the suppression of human colon cancer cells by promoting apoptosis. The impact of this treatment on cell viability, apoptosis, and the expression levels of Bcl‑2 family members, caspase family members and mitochondria‑related molecules in HT‑29 cells was assessed by the MTT assay, immunocytochemistry, western blotting and quantitative PCR. The combination of 5‑FU and α‑PA had a synergistic inhibitory effect on cell viability, as determined by assessing the combination index value. Bax protein expression levels were higher in the 50, 100 or 250 µM α‑PA combined with 5‑FU groups compared with those in the 5‑FU alone group (P<0.05). By contrast, Bcl‑2 protein expression levels and mitochondrial membrane potential (MMP, ΔΨm) were lower in the 100 or 250 µM α‑PA combined with 5‑FU groups than those in the 5‑FU alone group (P<0.05). In addition, hexokinase‑2 (HK‑2) protein expression levels were lower in the 50, 100 or 250 µM α‑PA combined with 5‑FU groups than those in the 5‑FU alone group (P<0.05). Compared with 5‑FU alone, after HT‑29 cells were treated with 50, 100 or 250 µM α‑PA combined with 5‑FU, the mRNA expression levels of extrinsic‑induced apoptotic molecules, including caspase‑8 and Bid, were higher (P<0.05). Treatment with 50, 100 or 250 µM α‑PA combined with 5‑FU also increased the mRNA expression levels of cytochrome c, caspase‑9 and caspase‑3, regulating intrinsic apoptosis (P<0.05). These results showed that α‑PA and 5‑FU had a synergistic effect on reducing the viability of human colon cancer HT‑29 cells by inducing extrinsic and intrinsic apoptosis pathways. The mechanism by which apoptosis is induced may involve the intrinsic apoptosis pathway that activates the mitochondria‑dependent pathway, including regulating the expression levels of Bcl‑2 family members, including Bax, Bcl‑2 and Bid, regulating MMP and HK‑2 expression levels, and increasing the expression of caspase cascade molecules, including caspase‑9 and caspase‑3. In addition, it may involve the extrinsic apoptosis pathway that activates caspase‑8 and caspase‑3 leading to apoptosis.

This study investigated the impact of long-term physical inactivity on hepatic cytoprotective- and inflammatory-related protein expressions in young rats and the subsequent apoptotic response during microgravity stress simulated by tail suspension. Four-week-old male Wistar rats were randomly assigned to the control (CT) and physical inactivity (IN) groups. The floor space of the cages provided to the IN group was reduced to half of that provided to the CT group. After 8 weeks, rats in both groups (n = 6-7) underwent tail suspension. Their livers were harvested immediately before (0 day) or 1, 3, and 7 days after tail suspension. Levels of hepatic heat shock protein 72 (HSP72), an anti-apoptotic protein, reduced over 7 days of tail suspension in the IN group than in the CT group (p < 0.01). Fragmented nucleosomes in the cytoplasmic fraction of the liver, an apoptotic index, were drastically increased by physical inactivity and tail suspension, and this change was significantly greater after 7 days of tail suspension in the IN group than in the CT group (p < 0.01). The apoptotic response was accompanied by the upregulation of pro-apoptotic proteins (cleaved caspase-3 and -7). Moreover, the levels of other pro-apoptotic proteins (tumor necrosis factor-1α and histone deacetylase 5) were also significantly higher in the IN than in the CT group (p < 0.05). Our results indicated that 8 weeks of physical inactivity decreased hepatic HSP72 levels and promoted hepatic apoptosis during the subsequent 7 days of tail suspension.

Proteolytic enzymes are one of the important biomarkers that enable the early diagnosis of several diseases, such as cancers. A specific proteolytic enzyme selectively degrades a certain sequence of a polypeptide. Therefore, a particular proteolytic enzyme can be selectively quantified by changing detectable signals causing degradation of the peptide chain. In addition, by combining polypeptides with various functional nanomaterials, proteolytic enzymes can be measured more sensitively and rapidly. In this paper, proteolytic enzymes that can be measured using a polypeptide degradation method are reviewed and recently studied functional nanomaterials-based proteolytic biosensors are discussed. We anticipate that the proteolytic nanobiosensors addressed in this review will provide valuable information on physiological changes from a cellular level for individual and early diagnosis.

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.

Neuroblastoma is the most common solid malignant tumor in infants and young children. Its origin is the incompletely committed precursor cells from the autonomic nervous system. Neuroblastoma cells are multipotent cells with a high potency of differentiation into the neural cell types. Neural differentiation leads to the treatment of neuroblastoma by halting the cell and tumor growth and consequently its expansion. Caspases are a family of proteins involved in apoptosis and differentiation. The present study aimed to investigate the potential role of caspase-9 activation on the differentiation of the human neuroblastoma SH-SY5Y cells. Here we investigated the caspase-9 and 3/7 activity during 1,25-dihydroxycholecalciferol (D3)-mediated differentiation of SH-SY5Y cells and took advantage of the inducible caspase-9 system in putting out the differentiation of the neuroblastoma cells. D3-induced differentiation of the cells could lead to activation of caspase-9 and caspase-3/7, astrocyte-like morphology, and increased expression of Glial fibrillary acidic protein (GFAP). By using the inducible caspase-9 system, we showed differentiation of SH-SY5Y cells to astrocyte-like morphology and increased level of GFAP expression. Furthered studies using a specific caspase-9 inhibitor showed inhibition of differentiation mediated by D3 or caspase-9 to astrocyte-like cells. These results show the potency of caspase-9 to direct differentiation of the human neuroblastoma SH-SY5Y cells into cells showing an astrocyte-like morphology.

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.

Carbon nanotubes (CNTs) have great potential as novel diagnostic or therapeutic tools in biomedicine but, cellular toxicity must be well considered before widespread application of CNTs. Many chemical agents exert their toxicity through apoptotic pathways by induction of caspase biomolecules. In the current study, effects of carboxyl-functionalized single-walled (SW) and multi-walled (MW) CNTs at a single dose of 100 µg ml-1 on the survival of Jurkat cells were examined using MTT assay. Additionally, the impacts of carboxylated CNTs on the gene expression levels of selected caspases were investigated. Jurkat cells were exposed to CNTs (100 µg ml-1 for 72 h) and then expression levels of selected caspase genes (Cas) were evaluated by qRT-PCR analysis. Housekeeping genes, β-actin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), were used as normalization controls. The results showed only a mild decrease in the viability of Jurkat cells treated with carboxylated MWCNT. The results of qRT-PCR analysis revealed the elevated level of Cas2 mRNA in the cells treated with carboxylated MWCNT (6.08-fold) and carboxylated SWCNT (1.20-fold). The expression levels of Cas4, Cas6, Cas8, and Cas10 genes were increased not significantly compared to the control untreated cells. Our findings suggested that exposure to carboxyl-functionalized CNTs could be resulted in up-regulation of the Cas2 gene and not initiator Cas8 and Cas10 genes. In addition, it seems that carboxylated MWCNT was more potent than SWCNT in activation of Cas2 gene expression and triggering cell death signal in a manner different from intrinsic or extrinsic apoptosis pathways.

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.