Human Lung Squamous Carcinoma Research Articles

Expression and prognosis analysis of TBX2 subfamily in human lung carcinoma.

Lung cancer has a high morbidity and mortality rate of all cancers worldwide. Therefore, there is an urgent need for reliable cancer markers for diagnosis and prognosis of patients with lung cancer. In this study, we used the bioinformatics database to compare the expression of the TBX2 subfamily at the transcriptional and protein levels in non-small cell lung cancer. Then, to confirm our bioinformatics analysis above, we used western bloting to determine the expression of TBX2, TBX3, TBX4 and TBX5 in human lung squamous carcinoma cell lines. Besides, low expression of TBX2 subfamily predicted a poor prognosis of patients with lung cancer. Finally, The methylation database was used to explore the relationship between the low expression of TBX2 subfamily and methylation of gene promoter region. Our data showed a significant decrease of TBX2 subfamily expression in lung cancer tissues of several histological subtypes. Finally, the methylation of TBX2 subfamily members in the promoter region of NSCLC was significantly higher than that in normal tissues. Our research provided sufficient evidence that TBX2 subfamily might play an inhibitory role in malignancy progression of lung cancer, which is promising to shed light on discovering a novel reliable cancer marker for prognosis of lung cancer patients.

Otilonium Bromide acts as a selective USP28 inhibitor and exhibits cytotoxic activity against multiple human cancer cell lines

USP28 contributes to tumorigenesis through modulating the lifespan of oncogenic factors such as c-Myc and ΔNp63, and it has been identified as a potential target for anti-cancer drug development. Currently, although quite a number of USP28 inhibitors have been developed, they all are still in preclinical research stage. Besides, none of them exhibits satisfying inhibition selectivity against USP28 over its closest homologue USP25. Here in this manuscript, a high-throughput screening aiming to discover USP28 inhibitors with novel scaffold and enhanced inhibition selectivity were conducted. After the primary screening and the second round of validation, Otilonium Bromide, an approved drug for treating irritable bowel syndrome, was identified to inhibit USP28′s activity with the IC50 value at 6.90 ± 0.90 μM. Besides, the drug exhibits a 3–4 folds inhibition selectivity against USP28 over USP25. According to the enzymatic kinetics analysis data and the hydrogen–deuterium exchange mass spectrometry results, Otilonium Bromide could bind to the allosteric pocket of USP28 and inhibit its activity in a reversible and non-competitive mode. The following performed cell-based assays revealed that the drug could cause cytotoxicity against human colorectal cancer cells and lung squamous carcinoma cells potentially through down-regulating USP28′s oncogenic substrates c-Myc and/or ΔNp63. Meanwhile, since Otilonium Bromide has been found to preferentially distribute to gastrointestinal tissues, we then evaluated its potential in the combination treatment of colorectal cancer cells with Regorafenib, which is an approved drug for colorectal cancer therapy. As expected, Otilonium Bromide could significantly enhance the sensitivity of colorectal cancer cells to Regorafenib.

Expression and Purification of FGFR1-Fc Fusion Protein and Its Effects on Human Lung Squamous Carcinoma.

Molecular-targeted therapies for lung squamous cell carcinoma (LSCC) are limited mainly because targetable oncogenic aberrations are absent in LSCC. Recent genomic analyses have revealed that the fibroblast growth factor (FGF) signaling pathway plays a fundamental role in LSCC progression via cancer cell proliferation and angiogenesis. In the present study, we designed, expressed, and purified a fibroblast growth factor receptor fragment (FGFR1-Fc) fusion protein using NS/0 cells. In FGF2-FGFR1 overexpressed NCI-H1703 cells, the FGFR1-Fc fusion protein effectively inhibited proliferation and invasion and arrested the cell cycle at the G0-G1 phase. In NCI-H1703 cells treated with the FGFR1-Fc fusion protein, the phosphorylation levels of FGFR1, FRS2, ERK, and AKT were significantly reduced. Using an siRNA assay, we demonstrated that FGF2-FGFR1 is the major anti-tumor target of FGFR1-Fc fusion the FGFR1-Fc fusion protein, which also significantly inhibited proliferation and invasion by NCI-H1703 cells via the FGF2-FGFR1 signaling pathway. In addition, the FGFR1-Fc fusion protein significantly inhibited angiogenesis in an embryonic chorioallantoic membrane model. The FGFR1-Fc fusion protein may be an effective therapeutic candidate for LSCC.

Structure-based discovery of potent USP28 inhibitors derived from Vismodegib

Ubiquitin-specific proteases (USPs) 28 is overexpressed in multiple types of cancers. The development of potent USP28 inhibitors is still in primitive stage. We previously reported our discovery of Vismodegib as a USP28 inhibitor by screening a commercially available drug library. Herein, we report our efforts to solve the cocrystal structure of Vismodegib bound to USP28 for the first time and subsequent structure-based optimization leading to a series of Vismodegib derivatives as potent USP28 inhibitors. Based on the cocrystal structure, elaborative SARs exploration was carried out to afford much more potent USP28 inhibitors than Vismodegib. The representative compounds 9l, 9o and 9p bearing high potency on USP28 showed high selectivity over USP2, USP7, USP8, USP9x, UCHL3 and UCHL5. The detailed cellular assay suggested that compounds 9l, 9o and 9p could cause cytotoxicity in both human colorectal cancer and lung squamous carcinoma cells and significantly enhance the sensitivity of colorectal cancer cells to Regorafenib. Further immunoblotting analysis indicated that compounds 9l, 9o and 9p could dose-dependently down-regulate the cellular level of c-Myc through ubiquitin-proteasome system and anti-cancer effects could mainly be attributed to their inhibition on USP28 but not involving the Hedgehog-Smoothened pathway. Thus, our work provided a series of novel and potent USP28 inhibitors derived from Vismodegib and may contribute to the development of USP28 inhibitors.

[Licochalcone A induces cell cycle arrest in human lung squamous carcinoma cells via the PI3K/Akt signaling pathway].

To investigate the effect of licochalcone A (LCA) on the proliferation and cell cycle of human lung squamous carcinoma cells and explore its possible molecular mechanism. MTT assay was used to detect the changes in proliferation of H226 cells after treatment with different concentrations of LCA for 48 h, and the IC50 of LCA was calculated. Flow cytometry was used to analyze cell cycle changes in H226 cells treated with 10, 20, and 40 μmol/L LCA, and the expressions of cyclin D1, cyclin-dependent kinase CDK2 and CDK4, and p-PI3K, PI3K, p-Akt, and Akt in the treated cells were detected using Western blotting. The effect of intraperitoneal injection of LCA for 24 days on tumor volume and weight was assessed in a BALB/c-nu mouse model bearing lung squamous carcinoma xenografts. MTT assay showed that LCA significantly decreased the viability of H226 cells with an IC50 of 28.3 μmol/L at 48 h. Flow cytometry suggested that LCA treatment induced obvious cell cycle arrest at the G1 phase. LCA treatment also significantly decreased the expressions of cyclin D1, CDK2, and CDK4, and inhibited the phosphorylation of PI3K and Akt in H226 cells. In the tumor-bearing mice, LCA treatment for 24 days significantly reduced the tumor volume and weight. LCA is capable of inhibiting the proliferation and inducing cell cycle arrest in lung squamous carcinoma cells possibility by regulating the PI3K/Akt singling pathway.

Heat shock protein 90 inhibitor 17-AAG down-regulates thymidine phosphorylase expression and potentiates the cytotoxic effect of tamoxifen and erlotinib in human lung squamous carcinoma cells

Elevated thymidine phosphorylase (TP) levels, a key enzyme in the pyrimidine nucleoside salvage pathway, in cancer cells, are related to a poor prognosis in a variety of cancers. Heat shock protein 90 (Hsp90) is a ubiquitous molecular chaperone that is involved in the stabilization and maturation of many oncogenic proteins. The aim of this study is to elucidate whether Hsp90 inhibitor 17-AAG could enhance tamoxifen- and erlotinib-induced cytotoxicity in nonsmall cell lung cancer (NSCLC) cells via modulating TP expression in two squamous NSCLC cell lines, H520 and H1703. We found that 17-AAG reduced TP expression via inactivating the MKK1/2-ERK1/2-mitogen-activated protein kinase (MAPK) pathway. TP knockdown with siRNA or ERK1/2 MAPK inactivation with the pharmacological inhibitor U0126 could enhance the cytotoxic and growth inhibitory effects of 17-AAG. In contrast, MKK1-CA or MKK2-CA (a constitutively active form of MKK1/2) vector-enforced expression could reduce the cytotoxic and cell growth inhibitory effects of 17-AAG. Furthermore, 17-AAG enhanced the cytotoxic and cell growth inhibitory effects of tamoxifen and erlotinib in NSCLC cells, which were associated with TP expression downregulation and MKK1/2-ERK1/2 signal inactivation. Taken together, Hsp90 inhibition downregulates TP, enhancing the tamoxifen- and erlotinib-induced cytotoxicity in H520 and H1703 cells.

Expression and Purification of FGFR1-Fc Fusion Protein, and its Effects on Human Lung Squamous Carcinoma

Expression and Purification of FGFR1-Fc Fusion Protein, and its Effects on Human Lung Squamous Carcinoma

Curcumin and isonicotinic acid hydrazide functionalized gold nanoparticles for selective anticancer action

Besides physicochemical properties, careful design of biomolecular surface corona can dictate therapeutic potential of nanoparticles. Therefore, we demonstrate a new approach to improve the anticancer potential of gold nanoparticles (AuNPs) by developing a strong corona of curcumin and isonicotinic acid hydrazide (INH) around them. Here, curcumin reduced AuNPsCur are surface-functionalized with biologically active INH (AuNPsCur@INH). Our investigations on human lung squamous carcinoma (LK-2) and human lung fibroblast (TIG-120) cells indicate selective toxicity of functionalized nanoparticles. Conjugation of curcumin and INH on the surface of AuNPs induced anticancer action through reactive oxygen species (ROS) generation. The further extension of this study revealed apoptosis and morphological changes in LK-2 and TIG-120 cells, respectively. Moreover, the anticancer potential of these nanoparticles is compared with standard cisplatin. The outcomes of the work determine that the facile functional surfaces on nanoparticles can be important in controlling the biological activities of nanoparticles.

[Microwave Hyperthermia Combined with Gemcitabine Inhibits Proliferation 
and Induces Apoptosis of Human Lung Squamous Carcinoma Cells].

背景与目的肺癌是全球发病率及死亡率最高的恶性肿瘤之一，寻找有效的抗肿瘤方法显得很重要，微波热疗作为一种新的治疗技术越来越受到重视。本研究旨在探讨微波热疗联合吉西他滨对人肺鳞癌细胞NCI-H1703和NCI-H2170体外增殖的影响及诱导凋亡的机制。方法CCK-8法分别检测微波热疗、吉西他滨对细胞增殖影响以及微波热疗与吉西他滨不同序贯方式对细胞增殖的影响；克隆形成实验观察阴性对照组、微波热疗组、吉西他滨组及热化联合组对细胞克隆形成的影响；流式细胞术检测不同处理组细胞的总凋亡率；Caspase-3、Caspase-8活性检测试验检测各组细胞Caspase-3、Caspase-8酶活性；CCK-8法检测AC-DEVD(Caspase-3抑制剂)对细胞增殖的影响；Western blot检测微波热疗和吉西他滨对肺鳞癌细胞凋亡相关蛋白的表达。结果微波热疗对肺鳞癌有增殖抑制作用；吉西他滨对两株细胞的IC50值分别为8.89 μmol/L和44.18 μmol/L；先化疗后微波热疗对两株肺鳞癌细胞有协同作用且可明显抑制细胞的克隆形成(P < 0.001)、促进细胞凋亡(P < 0.001)以及显著提高Caspase-3酶活性(P < 0.001)，但对Caspase-8酶活性影响不大(P > 0.05)；加入AC-DEVD(Caspase-3抑制剂)后，热化联合组细胞增殖率提高(P < 0.001)；Western blot检测显示微波热疗联合吉西他滨可上调p53、Caspase-3、Cleaved-Caspase-3、Cleaved-PARP以及Bax蛋白的表达。结论先吉西他滨后微波热疗可协同抑制人肺鳞癌细胞的体外增殖以及促进凋亡，其机制可能是通过活化p53、切割PARP蛋白诱导癌细胞发生Caspase-3依赖性凋亡，从而发挥其协同抑制癌细胞增殖的作用。

Insulin‑like growth factor‑1 receptor knockdown enhances radiosensitivity via the HIF‑1α pathway and attenuates ATM/H2AX/53BP1 DNA repair activation in human lung squamous carcinoma cells

Insulin-like growth factor-1 receptor (IGF-1R) is a cell membrane receptor involved in cell proliferation and apoptosis, which is highly expressed in lung squamous cell carcinoma (SCC). The present study aimed to observe the influence of IGF-1R silencing on the radiosensitivity of SCC and investigate the potential mechanisms involved. Human lung SCC H520 cells with relatively high expression of IGF-1R were used. IGF-1R expression was silenced using short hairpin RNA. The influence of IGF-1R silencing on radiosensitivity and apoptosis was assessed using a clone formation assay and flow cytometry. The expression levels of proteins relevant in DNA damage repair and hypoxic signaling pathways were analyzed using western blotting. Decreased expression of IGF-1R led to an increase in the sensitivity of H520 cells to irradiation. Molecular analysis showed that the reduced expression of IGF-1R decreased the protein expression of ataxia-telangiectasia mutated (ATM), H2A histone family member X (H2AX) and p53 binding protein 1 (53BP1), which are associated with the DNA repair pathway. Furthermore, 53BP1 is also known to be involved in apoptosis. Proteins involved in the hypoxic pathway, including hypoxia inducible factor 1 α (HIF-1α), matrix metallopeptidase 9 (MMP-9) and vascular endothelial growth factor A (VEGFA) were also involved in the radiosensitivity. In conclusion, decreased expression of IGF-1R leads to improved radiosensitivity of SCC cells, and the underlying mechanism may be associated with the decreased expression of proteins involved in ATM/H2AX/53BP1 DNA damage repair and the HIF-1α/MMP-9 hypoxic pathway, which results in the induction of apoptosis and increased radiosensitivity. These findings suggest that targeting of IGF-1R may represent a novel approach for lung SCC radiation treatment.

Targeting protein for Xenopus kinesin-like protein 2 knockdown enhances radiation sensitivity of human lung squamous carcinoma cell.

The targeting protein for Xenopus kinesin-like protein 2 (TPX2) has been demonstrated to be associated with the tumourigenesis of many cancers. In the present study, we investigated the role and preliminary mechanism of TPX2 in the resistance of lung squamous carcinoma to radiation therapy. The results showed that SK-MES-1R and NCI-H226R cells were more resistant to X-ray irradiation than the parental cells (SK-MES-1 and NCI-H226). Moreover, TPX2 was upregulated in the radioresistant cells compared with the parental cells. TPX2 knockdown significantly decreased TPX2 expression in SK-MES-1 cells, while TPX2 overexpression increased TPX2 expression in NCI-H226 cells compared with the corresponding control cells. TPX2 knockdown enhanced the radiosensitivity of SK-MES-1 and promoted cell apoptosis following exposure to irradiation, whereas TPX2 overexpression decreased the radiosensitivity of NCI-H226 and inhibited cell apoptosis. In in vivo studies, the combination of TPX2 knockdown and irradiation significantly inhibited tumour growth, decreased tumour weight, downregulated TPX2 expression in tumour tissue and induced cell apoptosis in nude mice, while TPX2 overexpression exerted an opposite effect. Our results indicated that TPX2 was correlated with cell radioresistance and it might be served as a therapeutic target to enhance cell radiosensitivity in the radiation therapy of lung squamous carcinoma.

Isoalantolactone induces intrinsic apoptosis through p53 signaling pathway in human lung squamous carcinoma cells.

Isoalantolactone has recently been revealed to induce apoptosis in several types of cancer. However, little is reported on its anti-tumor potential on human lung cancer. Our present study was designed to investigate its effects on human lung squamous carcinoma SK-MES-1 cells. We found that Isoalantolactone induced cellular and DNA morphological changes and decreased the viability of SK-MES-1 cells. It significantly inhibited the growth of SK-MES-1 cells through apoptosis in a dose-dependent manner via activation of p53. It also induced cell cycle arrest at G1 phase. It can down-regulate Bcl-2 and up-regulate Bax, to induce dissipation of mitochondrial membrane potential and generation of reactive oxygen species. Caspase-3 was also activated by Isoalantolactone, with the cleavage of poly (ADP-ribose) polymerase. Our results reveal that Isoalantolactone induces intrinsic apoptosis in SK-MES-1 cells through p53 signaling pathway, which suggests that Isoalantolactone could be a potential leading compound for future development of anti-lung cancer drugs.

Claudin-5, -7, and -18 suppress proliferation mediated by inhibition of phosphorylation of Akt in human lung squamous cell carcinoma

Abnormal expression of claudin (CLDN) subtypes has been reported in various solid cancers. However, it is unknown which subtype plays a key role in the regulation of proliferation in cancer cells. The expression of CLDN3-5, 7, and 18 in human lung squamous carcinoma tissues was lower than that in normal tissue. Here, we examined which combination of exogenous CLDNs expression inhibits proliferation and the molecular mechanism using human lung squamous RERF-LC-AI cells. Real-time polymerase chain reaction and western blotting showed that CLDN3-5, 7, and 18 are little expressed in RERF-LC-AI cells. In the exogenously transfected cells, CLDN5, 7, and 18 were distributed in the cell–cell contact areas concomitant with ZO-1, a tight junctional scaffolding protein, whereas CLDN3 and 4 were not. Cell proliferation was individually and additively suppressed by CLDN5, 7, and 18. The expression of these CLDNs showed no cytotoxicity compared with mock cells. CLDN5, 7, and 18 increased p21 and decreased cyclin D1, resulting in the suppression of cell cycle G1-S transition. The expression of these CLDNs inhibited phosphorylation of Akt without affecting phosphorylated ERK1/2. Furthermore, these CLDNs inhibited the nuclear localization of Akt and its association with 3-phosphoinositide-dependent protein kinase-1 (PDK1). The suppression of G1-S transition caused by CLDN5, 7, and 18 was rescued by the expression of constitutively active-Akt. We suggest that the reduction of CLDN5, 7, and 18 expression loses the suppressive ability of interaction between PDK1 and Akt and causes sustained phosphorylation of Akt, resulting in the disordered proliferation in lung squamous carcinoma cells.

Costunolide induces G1/S phase arrest and activates mitochondrial-mediated apoptotic pathways in SK-MES 1 human lung squamous carcinoma cells.

Despite the availability of several therapeutic options, a safer and more effective modality strategy is required for the treatment of lung cancer. Costunolide, a sesquiterpene lactone which isolated from the Saussurea lappa, has potent anticancer properties. In the present study, the effects of costunolide on cell viability, the cell cycle and apoptosis in SK-MES-1 human lung squamous carcinoma cells were investigated. Costunolide induced morphological changes and inhibited growth of SK-MES-1 cells growth. Flow cytometric analysis data demonstrated that costunolide significantly induced apoptosis of SK-MES-1 cells and induced cell cycle arrest at G1/S phase in a dose-dependent manner. Through upregulation in the expression of p53 and Bax, and downregulation in the expression of Bcl-2 and activation of caspase-3, costunolide-induced apoptosis was confirmed by western blot analysis. In addition, the significant loss of mitochondrial membrane potential indicated that costunolide may induce apoptosis via the mitochondria-dependent pathway in SK-MES-1 cells. These results highlight the potential effects of costunolide as an anti-cancer agent in a human lung squamous carcinoma cell line.

Dracorhodin perchlorate induces G1/G0 phase arrest and mitochondria-mediated apoptosis in SK-MES-1 human lung squamous carcinoma cells.

Dracorhodin perchlorate (DP) has recently been revealed to induce apoptosis in various types of cancer. However, the antitumor potential and molecular mechanisms of DP in human lung cancer have not been previously reported. Therefore, the present study aimed to investigate the effects of DP on cell viability, the cell cycle and apoptosis, using an MTT assay, flow cytometry and western blot studies. DP was identified to induce cellular and DNA morphological changes, and decreased the viability of SK-MES-1 human lung squamous carcinoma cells. DP significantly inhibited the growth of SK-MES-1 cells by inducing apoptosis and G1/G0 cell cycle arrest in a dose-dependent manner via activation of p53 (P<0.05). Furthermore, DP promoted the significant upregulation of B cell lymphoma-2 (Bcl-2)-activated X protein and significant downregulation of Bcl-2 (P<0.05), inducing dissipation of the mitochondrial membrane potential (MMP). In addition, caspase-3 was activated by DP via the cleavage of its substrate, proteolytic cleavage of poly(ADP-ribose) polymerase. DP also induced caspase-independent apoptosis by significantly increasing the protein expression of the apoptosis-inducing factor (P<0.05), which is localized in mitochondria under the physiological conditions and released into the cytoplasm when MMP is dissipated. Furthermore, the present study demonstrated that DP significantly increased the generation of reactive oxygen species (P<0.05). In conclusion, the current study revealed that DP is able to induce cell cycle arrest and apoptosis in SK-MES-1 cells via activation of the mitochondrial pathway, indicating that DP may be a potential leading compound for the development of future lung cancer therapeutic regimes.

Fructus phyllanthi tannin fraction induces apoptosis and inhibits migration and invasion of human lung squamous carcinoma cells in vitro via MAPK/MMP pathways.

Fructus phyllanthi tannin fraction (PTF) from the traditional Tibetan medicine Fructus phyllanthi has been found to inhibit lung and liver carcinoma in mice. In this study we investigated the anticancer mechanisms of PTF in human lung squamous carcinoma cells in vitro. Human lung squamous carcinoma cell line (NCI-H1703), human large-cell lung cancer cell line (NCI-H460), human lung adenocarcinoma cell line (A549) and human fibrosarcoma cell line (HT1080) were tested. Cell viability was detected with MTT assay. Cell migration and invasion were assessed using a wound healing assay and a transwell chemotaxis chambers assay, respectively. Cell apoptosis was analyzed with flow cytometric analysis. The levels of apoptosis-related and metastasis-related proteins were detected by Western blot and immunofluorescence. PTF dose-dependently inhibited the viability of the 3 human lung cancer cells. The IC50 values of PTF in inhibition of NCI-H1703, NCI-H460, and A549 cells were 33, 203, and 94 mg/L, respectively. PTF (15, 30, and 60 mg/L) dose-dependently induced apoptosis of NCI-H1703 cells. Treatment of NCI-H1703 and HT1080 cells with PTF significantly inhibited cell migration, and reduced the number of invasive cells through Matrigel. Furthermore, PTF dose-dependently down-regulated the expression of phosphor-ERK1/2, MMP-2 and MMP-9, up-regulated the expression of phosphor-JNK, but had no significant effect on the expression of ERK1/2 or JNK. PTF induces cell apoptosis and inhibits the migration and invasion of NCI-H1703 cells by decreasing MPPs expression through regulation of the MAPK pathway.

In vitro antiproliferative effect of trastuzumab (Herceptin(®)) combined with cetuximab (Erbitux(®)) in a model of human non-small cell lung cancer expressing EGFR and HER2.

Lung cancer is the leading cause of cancer death. For this reason, new therapies are needed for the treatment of this devastating disease. In this study, we investigated the effects of combining cetuximab and the trastuzumab on the growth of a model of human non-small cell lung carcinoma cell line (A549). The results were compared with those obtained from a human lung squamous carcinoma cell line (NCI-H226). Both cell lines were treated with cetuximab and trastuzumab, alone or in combination, at various concentrations, for 24, 48 and 72h. Cell proliferation was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. EGFR and HER-2 mRNA expression was detected by reverse transcription polymerase chain reaction, and the gene amplification status of receptors was evaluated by fluorescence in situ hybridisation. The colorimetric proliferation assay showed that trastuzumab combined with cetuximab significantly inhibited A549 cells at a dose of 40μg/ml after 72h of treatment (p<0.05), while no time-dose dependent inhibition was observed in NCI-H226 cells. The combined treatment influenced both levels of EGFR and HER-2 mRNA in A549 cells and only EGFR mRNA levels in NCI-H226 cells. Fluorescence in situ hybridisation showed that both cell lines were aneuploid for the two genes with equally increased EGFR and CEN7 signals, as well as HER-2 and CEN17 signals, indicating a condition of polysomy without amplification. The preliminary results of this study encourage further investigations to elucidate the downstream events involved and to understand how these mechanisms influence non-small cell lung cancers growth.

Effects of Cx43 gene modification on the proliferation and migration of the human lung squamous carcinoma cell line NCI-H226.

In this study, the human lung squamous carcinoma cell line NCI-H226 was transfected with the recombinant plasmid pBudCE4.1_Cx43 to explore the role of the Cx43 gene in cell growth, cell cycle, and tumor migration. pBudCE4.1-Cx43 was transfected into human lung squamous carcinoma NCI-H226 cells using Lipofectamine TM2000. The mRNA and protein expressions of Cx43 in the transfected cells were detected by reverse transcriptase polymerase chain reaction and western blot analysis. The cell-cell communication was detected using the scratch dye tracer method and the cell cycle was detected by flow cytometry. The CCK-8 proliferation, scratch healing, and cell invasion assays were performed to evaluate the effect of the Cx43 gene transfection on the proliferation, migration, and invasive abilities of NCI-H226 cells. Cx43 mRNA and protein expressions and the fluorescence intensity in the scratch healing test were significantly higher in the experimental group than those in the control and blank groups (P < 0.05 and < 0.01, respectively). The CCK-8 proliferation assay and the scratch healing experiment revealed significantly inhibited NCI-H226 cell proliferation (especially 72 h after incubation) and cell migration, respectively, in the experimental group, compared to the control and blank groups (P < 0.001 and <0.05, respectively). The transwell chamber test showed a statistically significant decrease in the invasive ability of NCI-H226 cells in the experimental group (P < 0.05). Therefore, Cx43 gene transfection could inhibit the migration of human lung squamous carcinoma cell line NCI-H226, thereby inhibiting tumor cell proliferation.

SCYL1BP1 has tumor-suppressive functions in human lung squamous carcinoma cells by regulating degradation of MDM2.

SCY1-like 1-binding protein 1 (SCYL1BP1) is a newly identified transcriptional activator domain containing protein with many unknown biological functions. Recently emerging evidence has revealed that it is a novel regulator of the p53 pathway, which is very important for the development of human cancer. However, the effects of SCYL1BP1 on human lung squamous carcinoma cell biological behavior remain poorly understood. In this study, we present evidence that SCYL1BP1 can promote the degradation of MDM2 protein and further inhibit the G1/S transition of lung squamous carcinoma cell lines. Functional assays found that reintroduction of SCYL1BP1 into lung squamous carcinoma cell lines significantly inhibited cell proliferation, migration, invasion and tumor formation in nude mice, suggesting strong tumor suppressive function of SCYL1BP1 in lung squamous carcinoma. Taken together, our data suggest that the interaction of SCYL1BP1/MDM2 could accelerate MDM2 degradation, and may function as an important tumor suppressor in lung squamous carcinomas.

Crotoxin induces apoptosis and autophagy in human lung carcinoma cells in vitro via activation of the p38MAPK signaling pathway.

Crotoxin (CrTX) is the primary toxin in South American rattlesnake (Crotalus durissus terrificus) venom, and exhibits antitumor and other pharmacological actions in vivo and in vitro. Here, we investigated the molecular mechanisms of the antitumor action of CrTX in human lung carcinoma cells in vitro. Human lung squamous carcinoma SK-MES-1 cells were tested. The cytotoxicity of CrTX was evaluated in both MTT and colony formation assays. Cell cycle was investigated with flow cytometry. Cell apoptosis was studied with Hoechst 33258 and Annexin V-FITC staining. The levels of relevant proteins were analyzed using Western blot assays. CrTX (25, 50, 100 μmol/L) inhibited the growth and colony formation of SK-MES-1 cells in dose- and time-dependent manners. CrTX increased the proportion of S phase cells and dose-dependently induced cell apoptosis, accompanied by down-regulating the expression of proliferating cell nuclear antigen (PCNA), and increasing the level of cleaved caspase-3. Furthermore, CrTX dose-dependently increased the expression of autophagy-related proteins LC3-II and beclin 1, and decreased the level of p62 in the cells. Moreover, CrTX (50 μmol/L) significantly increased p38MAPK phosphorylation in the cells. Pretreatment of the cells with SB203580, a specific inhibitor of p38MAPK, blocked the inhibition of CrTX on cell proliferation, as well as CrTX-induced apoptosis and cleaved caspase-3 expression. The p38MAPK signaling pathway mediates CrTX-induced apoptosis and autophagy of human lung carcinoma SK-MES-1 cells in vitro.