Evaluation of Laparoscopic Colorectal Resection Among Elderly Individuals With Colorectal Malignancy: A Single-center Retrospective Analysis.

OBJECTIVE: The purpose of the current study was to statistically clarify the precise risk age in elderly patients undergoing colorectal surgery and to evaluate the safety and efficacy of laparoscopic colorectal resection in these patients. METHODS: Patients' clinical variables were extracted from the database of the Gastrointestinal Surgery Centre, Third Affiliated Hospital of Sun Yat-sen University, from 2015 to 2019. Logistic regression was conducted to identify independent risk factors of postoperative complications and ORs for each age. Curves of odds ratios (ORs) and CIs for each age were fitted by using a locally weighted scatterplot smoother, and a structural breakpoint was determined by the Chow test to identify a precise cutoff risk age for elderly patients. Comparison and subgroup analysis were conducted between surgical approach groups using the Student t test and χ 2 analysis. RESULTS: Locally weighted scatterplot smoother OR analysis manifested that patients aged 69 years old or older suffered a higher possibility of postoperative complications and should be defined as high-risk age. Comparison according to the high-risk age revealed laparoscopic colorectal surgery is better than laparotomic surgery for elderly individuals in terms of hospital stay (9.46 ± 5.96 vs 15.01 ± 6.34, P < 0.05), the incidence of intensive care unit transfer (4 vs 20, P < 0.05), and incidence of surgical site infection (15 vs 20, P < 0.05). Patients who underwent laparotomic surgery had a greater prevalence of Clavien-Dindo II/III complications ( P < 0.05). These findings remained stable even after propensity matching. Furthermore, such superiority was proved especially significant for patients who underwent left-side colorectal resection. In addition, overall survival was improved in the laparoscopic surgery group, whereas no differences were observed in disease-free survival. CONCLUSION: In our study population, age 69 or older was a cutoff point age suggests a higher possibility of postoperative morbidity after colorectal surgery. Laparoscopic colorectal resection should be regarded as a superior therapeutic choice for these elderly individuals, especially for left-side colorectal surgeries.

Newcastle disease virus suppresses antigen presentation via inhibiting IL-12 expression in dendritic cells. / æ–°åŸŽç–«ç— æ¯’é€šè¿‡æŠ‘åˆ¶æ ‘çªçŠ¶ç»†èƒžç™½ä»‹ç´ 12的表达抑制抗原递呈.

As a potential vectored vaccine, Newcastle disease virus (NDV) has been subject to various studies for vaccine development, while relatively little research has outlined the immunomodulatory effect of the virus in antigen presentation. To elucidate the key inhibitory factor in regulating the interaction of infected dendritic cells (DCs) and T cells, DCs were pretreated with the NDV vaccine strain LaSota as an inhibitor and stimulated with lipopolysaccharide (LPS) for further detection by enzyme-linked immunosorbent assay (ELISA), flow cytometry, immunoblotting, and quantitative real-time polymerase chain reaction (qRT-PCR). The results revealed that NDV infection resulted in the inhibition of interleukin (IL)-12p40 in DCs through a p38 mitogen-activated protein kinase (MAPK)|-dependent manner, thus inhibiting the synthesis of IL-12p70, leading to the reduction in T cell proliferation and the secretion of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and IL-6 induced by DCs. Consequently, downregulated cytokines accelerated the infection and viral transmission from DCs to T cells. Furthermore, several other strains of NDV also exhibited inhibitory activity. The current study reveals that NDV can modulate the intensity of the innate|‒|adaptive immune cell crosstalk critically toward viral invasion improvement, highlighting a novel mechanism of virus-induced immunosuppression and providing new perspectives on the improvement of NDV-vectored vaccine.

Nanodrug modified with engineered cell membrane targets CDKs to activate aPD-L1 immunotherapy against liver metastasis of immune-desert colon cancer.

Immunotherapy based on the PD-1/PD-L1 axis blockade has no benefit for patients diagnosed with colon cancer liver metastasis (CCLM) for the microsatellite stable/proficient mismatch repair (MSS/pMMR)) subtype, which is known as an immune-desert cancer featuring poor immunogenicity and insufficient CD8+ T cell infiltration in the tumor microenvironment. Here, a multifunctional nanodrug carrying a cyclin-dependent kinase (CDK)1/2/5/9 inhibitor and PD-L1 antibody is prepared to boost the immune checkpoint blockade (ICB)-based immunotherapy against MSS/pMMR CCLM via reversing the immunosuppressive tumor microenvironment. To enhance the MSS/pMMR CCLM-targeting efficacy, we modify the nanodrug with PD-L1 knockout cell membrane of this colon cancer subtype. First, CDKs inhibitor delivered by nanodrug down-regulates phosphorylated retinoblastoma and phosphorylated RNA polymerase II and meanwhile arrests the G2/M cell cycle in CCLM to promote immunogenic signal release, stimulate dendritic cell maturation, and enhance CD8+ T cell infiltration. Moreover, CDKi suppresses the secretion of immunosuppressive cytokines in tumor-associated myeloid cells sensitizing ICB therapy in CCLM. Notably, the great efficacy to activate immune responses is demonstrated in the patient-derived xenograft model and the patient-derived organoid model as well, revealing a clinical application potential. Overall, our study represents a promising therapeutic approach for targeting liver metastasis, remolding the tumor immune microenvironment (TIME), and enhancing the response of MSS/pMMR CCLM to boost ICB immunotherapy.

AGCM-22, a novel cetuximab-based EGFR-targeting antibody-drug-conjugate with highly selective anti-glioblastoma efficacy.

The epidermal growth factor receptor (EGFR) has received significant attention as a potential target for glioblastoma (GBM) therapeutics in the past two decades. However, although cetuximab, an antibody that specifically targets EGFR, exhibits a high affinity for EGFR, it has not yet been applied in the treatment of GBM. Antibody-drug conjugates (ADCs) utilize tumor-targeting antibodies for the selective delivery of cytotoxic drugs, resulting in improved efficacy compared to conventional chemotherapy drugs. However, the effectiveness of cetuximab as a targeted antibody for ADCs in the treatment of GBM remains uncertain. In this study, we synthesized AGCM-22, an EGFR-targeted ADC derived from cetuximab, by conjugating it with the tubulin inhibitor monomethyl auristatin E (MMAE) using our Valine-Alanine Cathepsin B cleavable linker. In vitro experiments demonstrated that AGCM-22 effectively inhibited GBM cell proliferation through increased levels of apoptosis and autophagy-related cell death, whereas cetuximab alone had no anti-GBM effects. Additionally, both mouse and human orthotopic tumor models exhibited the selective tumor-targeting efficacy of AGCM-22, along with favorable metabolic properties and superior anti-GBM activity compared to temozolomide (TMZ). In summary, this study presents a novel ADC for GBM therapy that utilizes cetuximab as the tumor-targeting antibody, resulting in effective delivery of the cytotoxic drug payload.

Platycodin D2 enhances P21/CyclinA2-mediated senescence of HCC cells by regulating NIX-induced mitophagy.

BACKGROUND: Hepatocellular carcinoma (HCC) cells usually show strong resistance to chemotherapy, which not only reduces the efficacy of chemotherapy but also increases the side effects. Regulation of autophagy plays an important role in tumor treatment. Cell senescence is also an important anti-cancer mechanism, which has become an important target for tumor treatment. Therefore, it is of great clinical significance to find anti-HCC drugs that act through this new mechanism. Platycodin D2 (PD2) is a new saponin compound extracted from the traditional Chinese medicine Platycodon grandiflorum. PURPOSE: Our study aimed to explore the effects of PD2 on HCC and identify the underlying mechanisms. METHODS: First, the CCK8 assay was used to detect the inhibitory effect of PD2 on HCC cells. Then, different pathways of programmed cell death and cell cycle regulators were measured. In addition, we assessed the effects of PD2 on the autophagy and senescence of HCC cells by flow cytometry, immunofluorescence staining, and Western blotting. Finally, we studied the in vivo effect of PD2 on HCC cells by using a mouse tumor-bearing model. RESULTS: Studies have shown that PD2 has a good anti-tumor effect, but the specific molecular mechanism has not been clarified. In this study, we found that PD2 has no obvious toxic effect on normal hepatocytes, but it can significantly inhibit the proliferation of HCC cells, induce mitochondrial dysfunction, enhance autophagy and cell senescence, upregulate NIX and P21, and downregulate CyclinA2. Gene silencing and overexpression indicated that PD2 induced mitophagy in HCC cells through NIX, thereby activating the P21/CyclinA2 pathway and promoting cell senescence. CONCLUSIONS: These results indicate that PD2 induces HCC cell death through autophagy and aging. Our findings provide a new strategy for treating HCC.

Licochalcone A Induces Ferroptosis in Hepatocellular Carcinoma via Reactive Oxygen Species Activated by the SLC7A11/GPX4 Pathway.

Liver cancer is a common malignant tumor, and its incidence is increasing yearly. Millions of people suffer from liver cancer annually, which has a serious impact on global public health security. Licochalcone A (Lico A), an important component of the traditional Chinese herb licorice, is a natural small molecule drug with multiple pharmacological activities. In this study, we evaluated the inhibitory effects of Lico A on hepatocellular carcinoma cell lines (HepG2 and Huh-7), and explored the inhibitory mechanism of Lico A on hepatocellular carcinoma. First, we evaluated the inhibitory effects of Lico A on hepatocellular carcinoma, and showed that Lico A significantly inhibited and killed HepG2 and Huh-7 cells in vivo and in vitro. Transcriptomic analysis showed that Lico A inhibited the expression of solute carrier family 7 member 11 (SLC7A11), which induced ferroptosis. We confirmed through in vivo and in vitro experiments that Lico A promoted ferroptosis in hepatocellular carcinoma cells by downregulating SLC7A11 expression, thereby inhibiting the glutathione (GSH)-glutathione peroxidase 4 (GPX4) pathway and inducing activation of reactive oxygen species (ROS). In this study, we suggest that Lico A is a potential SLC7A11 inhibitor that induces ferroptotic death in hepatocellular carcinoma cells, thereby providing a theoretical basis for the development of natural small molecule drugs against hepatocellular carcinoma.

Identification of a small-molecule Tim-3 inhibitor to potentiate T cell-mediated antitumor immunotherapy in preclinical mouse models.

T cell immunoglobulin and mucin-containing molecule 3 (Tim-3), expressed in dysfunctional and exhausted T cells, has been widely acknowledged as a promising immune checkpoint target for tumor immunotherapy. Here, using a strategy combining virtual and functional screening, we identified a compound named ML-T7 that targets the FG-CC' cleft of Tim-3, a highly conserved binding site of phosphatidylserine (PtdSer) and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). ML-T7 enhanced the survival and antitumor activity of primary CD8+ cytotoxic T lymphocytes (CTLs) and human chimeric antigen receptor (CAR) T cells and reduced their exhaustion in vitro and in vivo. In addition, ML-T7 promoted NK cells' killing activity and DC antigen-presenting capacity, consistent with the reported activity of Tim-3. ML-T7 strengthened DCs' functions through both Tim-3 and Tim-4, which is consistent with the fact that Tim-4 contains a similar FG-CC' loop. Intraperitoneal dosing of ML-T7 showed comparable tumor inhibitory effects to the Tim-3 blocking antibody. ML-T7 reduced syngeneic tumor progression in both wild-type and Tim-3 humanized mice and alleviated the immunosuppressive microenvironment. Furthermore, combined ML-T7 and anti-PD-1 therapy had greater therapeutic efficacy than monotherapy in mice, supporting further development of ML-T7 for tumor immunotherapy. Our study demonstrates a potential small molecule for selectively blocking Tim-3 and warrants further study.

A novel antibody-KSP inhibitor conjugate improves KSP inhibitor efficacy in vitro and in vivo.

Many clinical trials of kinesin spindle protein (KSP) inhibitors have failed due to issues such as high toxicity and a short circulation half-life in vivo. To address the limitations of current KSP inhibitors and thus broad its use in antitumor therapy, this study applied antibody-drug conjugate (ADC) technology to the KSP inhibitor SB-743921, which was coupled with the HER2-specific antibody trastuzumab using a cathepsin B-dependent valine-alanine (Val-Ala, VA) dipeptide-type linker to generate H2-921. Ex vivo and in vivo analyses of H2-921 showed an increased half-life of SB-743921 and prolonged contact time with tumor cells. Furthermore, H2-921 induced apoptosis and incomplete autophagy in HER2-positive cells. In the in vivo analyses, H2-921 had significant tumor-targeting properties, and tumor inhibition by H2-921 was greater than that by traditional KSP inhibitors but similar to that by the positive control drug T-DM1. In conclusion, this study describes a novel application of ADC technology that enhances the antitumor effects of a KSP inhibitor and thus may effectively address the poor clinical efficacy of KSP inhibitors.

Human adenovirus type 7 subunit vaccine induces dendritic cell maturation through the TLR4/NF-κB pathway is highly immunogenic.

Introduction: Human adenovirus type 7 (HAdv-7) infection is the main cause of upper respiratory tract infection, bronchitis and pneumonia in children. At present, there are no anti- adenovirus drugs or preventive vaccines in the market. Therefore, it is necessary to develop a safe and effective anti-adenovirus type 7 vaccine. Methods: In this study, In this study, we used the baculovirus-insect cell expression system to design a recombinant subunit vaccine expressing adenovirus type 7 hexon protein (rBV-hexon) to induce high-level humoral and cellular immune responses. To evaluate the effectiveness of the vaccine, we first detected the expression of molecular markers on the surface of antigen presenting cells and the secretion of proinflammatory cytokines in vitro. We then measured the levels of neutralizing antibodies and T cell activation in vivo. Results: The results showed that the rBV-hexon recombinant subunit vaccine could promote DC maturation and improve its antigen uptake capability, including the TLR4/NF-κB pathway which upregulated the expression of MHCI, CD80, CD86 and cytokines. The vaccine also triggered a strong neutralizing antibody and cellular immune response, and activated T lymphocytes. Discussion: Therefore, the recombinant subunit vaccine rBV-hexon promoted promotes humoral and cellular immune responses, thereby has the potential to become a vaccine against HAdv-7.

Neobavaisoflavone induces pyroptosis of liver cancer cells via Tom20 sensing the activated ROS signal.

BACKGROUND: Neobavaisoflavone (NBIF), a natural active ingredient isolated from Psoralea, possesses anti-inflammatory, anti-cancer, and antioxidant properties; however, the anti-tumor mechanism of NBIF has not been thoroughly investigated, and the inhibitory effect and inhibitory pathway of NBIF on liver cancer are still unknown. PURPOSE: Our study aimed to explore the effects of NBIF on hepatocellular carcinoma and its potential mechanisms. METHODS: First, we detected the inhibition of NBIF on HCC cells by the CCK8 assay and then observed the morphological changes of the cells under the microscope. Besides, we analyzed the changes in the pyroptosis level of NBIF when inhibiting the cells through flow cytometry, immunofluorescence, and a western blot assay. Finally, we used a mouse tumor-bearing model to explore the effects of NBIF in vivo on HCCLM3 cells. RESULTS: NBIF-treated HCC cells exhibited specific features of pyroptosis. Analysis of pyroptosis-related protein levels revealed that NBIF primarily induced pyroptosis in HCC cells via the caspase-3-GSDME signaling pathway. Then, we demonstrated that NBIF impacted the protein expression of Tom20 by producing ROS in HCC cells, hence promoting the recruitment of Bax to mitochondria, activating caspase-3, cutting GSDME, and triggering pyroptosis. CONCLUSIONS: By activating ROS, NBIF was able to trigger pyroptosis in HCC cells, providing an experimental basis for the future study of new treatments for liver cancer.

Polygalacin D inhibits the growth of hepatocellular carcinoma cells through BNIP3L-mediated mitophagy and endogenous apoptosis pathways.

Platycodon grandiflorum (Jacq.) A. DC. is a famous medicinal plant commonly used in East Asia. Triterpene saponins isolated from P. grandiflorum are the main biologically active compounds, among which polygalacin D (PGD) has been reported to be an anti-tumor agent. However, its anti-tumor mechanism against hepatocellular carcinoma is unknown. This study aimed to explore the inhibitory effect of PGD in hepatocellular carcinoma cells and related mechanisms of action. We found that PGD exerted significant inhibitory effect on hepatocellular carcinoma cells through apoptosis and autophagy. Analysis of the expression of apoptosis-related proteins and autophagy-related proteins revealed that this phenomenon was attributed to the mitochondrial apoptosis and mitophagy pathways. Subsequently, using specific inhibitors, we found that apoptosis and autophagy had mutually reinforcing effects. In addition, further analysis of autophagy showed that PGD induced mitophagy by increasing BCL2 interacting protein 3 like (BNIP3L) levels.In vivo experiments demonstrated that PGD significantly inhibited tumor growth and increased the levels of apoptosis and autophagy in tumors. Overall, our findings showed that PGD induced cell death of hepatocellular carcinoma cells primarily through mitochondrial apoptosis and mitophagy pathways. Therefore, PGD can be used as an apoptosis and autophagy agonist in the research and development of antitumor agents.

Inhibitory Effects of Esculetin on Liver Cancer Through Triggering NCOA4 Pathway-Mediation Ferritinophagy in vivo and in vitro.

Objective: To explore the effects of Esculetin on liver cancer and explore potential mechanisms of Esculetin-inducing cells death. Methods: Esculetin's effects on the proliferation, migration and apoptosis of HUH7 and HCCLM3 cells were detected by using CCK8, crystal violet staining, wound healing, TranswellTM and Annexin V-FITC/PI. Flow cytometry, fluorescence staining, Western blot, T-AOC, DPPH radical scavenging assay, hydroxyl radical's inhibitory capability and GSH test were used to examine the esculetin's effects on the ROS level, the oxidation-related substances and proteins' expression in hepatoma cells. In vivo experiment was performed by xenograft model. Ferrostatin-1 was used to determine the death way of hepatoma cells induced by esculetin. Live cell probe, Western blot, Fe2+ content, MDA, HE staining, Prussian blue staining and immunohistochemistry were used to examine the ferritinophagy-related phenomenon induced by esculetin in hepatoma cells. The relationship between esculetin and NCOA4-mediated ferritinophagy was confirmed through gene silence and overexpression, immunofluorescence staining and Western blot. Results: Esculetin suppressed the proliferation, migration and apoptosis of HUH7 and HCCLM3 cells significantly, influenced the oxidative stress level, altered the autophagy and iron metabolism levels in cells, and produced a ferritinophagy-related phenomena. Esculetin increased the levels of cellular lipid peroxidation and reactive oxygen species. In vivo, esculetin could decrease tumour volume, promote LC3 and NCOA4 expressions, suppresse hydroxyl radical's inhibiting capacity and GSH, increase Fe2+ and MDA levels, decrease antioxidant proteins expression in tumour tissue. In addition, Esculetin could also increase the iron deposition of tumour tissues, promote ferritinophagy, and induce tumours' ferroptosis. Conclusion: Esculetin has an inhibitory effect on liver cancer in vivo and in vitro through triggering NCOA4 pathway-mediation ferritinophagy.

Human adenovirus type 7 virus-like particle vaccine induces Dendritic cell maturation through the TLR4/NF-κB pathway and is highly immunogenic.

Human adenovirus type 7 (HAdv-7) infection is the main cause of upper respiratory tract infection, bronchitis and pneumonia in children. At present, there are no anti-adenovirus drugs or preventive vaccines in the market. Therefore, it is necessary to develop a safe and effective anti-adenovirus type 7 vaccine. In this study, we designed a virus-like particle vaccine expressing the epitopes of hexon and penton of adenovirus type 7 with hepatitis B core protein (HBc) as the vector to induce high-level humoral and cellular immune responses. To evaluate the effectiveness of the vaccine, we first detected the expression of molecular markers on the surface of antigen presenting cells and the secretion of proinflammatory cytokines in vitro. We then measured the levels of neutralizing antibodies and T cell activation in vivo. The results showed that the HAdv-7 virus-like particles (VLPs) recombinant subunit vaccine could activate the innate immune response, including the TLR4/NF-κB pathway which upregulated the expression of MHC II, CD80, CD86, CD40 and cytokines. The vaccine also triggered a strong neutralizing antibody and cellular immune response and activated T lymphocytes. Therefore, the HAdv-7 VLPs promoted humoral and cellular immune responses, thereby potentially enhancing protection against HAdv-7 infection.

Apoptotic and autophagic cell death induced in cervical cancer cells by a dual specific oncolytic adenovirus.

OBJECTIVE: Oncolytic adenoviruses are capable of exerting anticancer effects via a variety of mechanisms, including apoptosis and autophagy. In the present study, the dual-specific antitumor oncolytic adenovirus, Ad-Apoptin-hTERT-E1a (ATV), was used to infect cervical cancer cell lines to test its antitumor effects. METHODS: To explore the use of apoptin in tumor gene therapy, a recombinant adenovirus ATV expressing the apoptin protein was assessed to determine its lethal and growth-inhibitory effects on human cervical cancer cell line (HeLa) cells in vitro . Nonapoptotic autophagy of HeLa cells infected with ATV was assessed by examining the cell morphology, development of acidic vesicular organelles and the conversion of microtubule-associated protein 1 light chain 3 (LC3) from its cytoplasmic to autophagosomal membrane form. Using gene silencing (knockdown of LC3 and Belin-1), autophagy-associated molecules (e.g. ATG5, ATG12 and ULK1) were monitored by real-time PCR and western blot. RESULTS: A series of experiments demonstrated that ATV could significantly induce apoptosis and autophagy in cervical cancer cells, and provided evidence that ATV not only induced apoptosis but also autophagy and ATG5, ATG12 and ULK1 related pathways were not entirely dependent on LC3 and Beclin-1. CONCLUSION: These results indicate that ATV may have a potential application in tumor gene therapy.

Apoptin causes apoptosis in HepG-2 cells via Ca2+ imbalance and activation of the mitochondrial apoptotic pathway.

BACKGROUND: Apoptin is derived from the chicken anemia virus and exhibits specific cytotoxic effects against tumor cells. Herein, we found that Apoptin induced a strong and lasting endoplasmic reticulum (ER) stress response, Ca2+ imbalance, and triggered the mitochondrial apoptotic pathway. The aim of this study was to explore the mechanisms by which Apoptin exhibited anti-tumor effects in HepG-2 cells. METHODS: The intracellular levels of calcium (Ca2+ ) were induced by ER stress and determined by electron microscopy, flow cytometry, and fluorescence staining. The mitochondrial injury was determined by mitochondrial membrane potential and electron microscopy. Western blotting was used to investigate the levels of key proteins in ER stress and the apoptotic pathway in mitochondria. The relationship between Ca2+ levels and apoptosis in Apoptin-treated cells was analyzed using a Ca2+ chelator (BAPTA-AM), flow cytometry, and fluorescence staining. We also investigated the in vivo effects of Ca2+ imbalance on the mitochondrial apoptotic pathway using tumor tissues xenografted on nude mice. RESULTS: This study showed that Apoptin induced a strong and long- lasting ER stress and injury, which subsequently led to an imbalance of cellular Ca2+ levels, a reduction in the mitochondrial membrane potential, a significant extent image in the mitochondrial structure, and an increase in the expression levels of Smac/Diablo and Cyto-C. CONCLUSIONS: In summary, Apoptin induced apoptosis in HepG-2 cells via Ca2+ imbalance and activation of the mitochondrial apoptotic pathway. This study provided a new direction for antitumor research in Apoptin.

Apoptin inhibits glycolysis and regulates autophagy by targeting pyruvate kinase M2 (PKM2) in lung cancer A549 cells.

Pyruvate kinase M2 (PKM2) is a key enzyme in aerobic glycolysis, and which plays an important role in tumor energy metabolism and tumor growth. Ad-apoptin, a recombinant oncolytic adenovirus, that can stably express apoptin in tumor cells and selectively causes cell death in tumor cells. The relationship between the anti-tumor function of apoptin, including apoptosis and autophagy activation, and energy metabolism of tumor cells has not been clarified. In this study, we used the A549 lung cancer cell line to analyze the mechanism of PKM2 involvement apoptin-mediated cell death in tumor cells. PKM2 expression in lung cancer cells was detected by Western blot and qRT-PCR. In the PKM2 knockdown and over-expression experiments, A549 lung cancer cells were treated with Ad-apoptin, and cell viability was determined by the CCK-8 assay and crystal violet staining. Glycolysis was investigated using glucose consumption and lactate production experiments. Moreover, the effects of Ad-apoptin on autophagy and apoptosis were analyzed by immunofluorescence using the Annexin v-mCherry staining and by western blot for c-PARP, p62 and LC3-II proteins. Immunoprecipitation analysis was used to investigate the interaction between apoptin and PKM2. In addition, following PKM2 knockdown and overexpression, the expression levels of p-AMPK, p-mTOR, p-ULK1, and p-4E-BP1 proteins in Ad-apoptin treated tumor cells, were analyzed by western blot to investigate the mechanism of apoptin effect on the energy metabolism of tumor cells. The in vivo antitumor mechanism of apoptin was analyzed by xenograft tumor inhibition experiment in nude mice and immunohistochemistry of tumors' tissue. As a result, apoptin could target PKM2, inhibit glycolysis and cell proliferation in A549 cells, and promote autophagy and apoptosis in A549 cells by regulating the PKM2/AMPK/mTOR pathway. This study confirmed the necessary role of Ad-apoptin in energy metabolism of A549 cells.

Apoptin mediates mitophagy and endogenous apoptosis by regulating the level of ROS in hepatocellular carcinoma.

BACKGROUND: Apoptin, as a tumor-specific pro-apoptotic protein, plays an important anti-tumoral role, but its mechanism of autophagy activation and the interaction between autophagy and apoptosis have not been accurately elucidated. Here, we studied the mechanism of apoptin-induced apoptosis and autophagy and the interaction between two processes. METHODS: Using crystal violet staining and the CCK-8 assay, we analyzed the effect of apoptin in the inhibition of liver cancer cells in vitro and analyzed the effect of inhibiting liver cancer in vivo by establishing a nude mouse tumor model. Flow cytometry and fluorescence staining were used to analyze the main types of apoptin-induced apoptosis and autophagy. Subsequently, the relationship between the two events was also analyzed. Flow cytometry was used to analyze the effect of ROS on apoptin-mediated apoptosis and autophagy mediated by apoptin. The effect of ROS on two phenomena was analyzed. Finally, the role of key genes involved in autophagy was analyzed using gene silencing. RESULTS: The results showed that apoptin can significantly increase the apoptosis and autophagy of liver cancer cells, and that apoptin can cause mitophagy through the increase in the expression of NIX protein. Apoptin can also significantly increase the level of cellular ROS, involved in apoptin-mediated autophagy and apoptosis of liver cancer cells. The change of ROS may be a key factor causing apoptosis and autophagy. CONCLUSION: The above results indicate that the increase in ROS levels after apoptin treatment of liver cancer cells leads to the loss of mitochondrial transmembrane potential, resulting in endogenous apoptosis and mitophagy through the recruitment of NIX. Therefore, ROS may be a key factor connecting endogenous apoptosis and autophagy induced by apoptin in liver cancer cells. Video abstract.

A comparative study of the ability of recombinant oncolytic adenovirus, doxorubicin and tamoxifen to inhibit the proliferation of breast cancer cells.

In this study, we compared the inhibitory effects of recombinant oncolytic adenovirus (Ad-apoptin-hTERTp-E1a, Ad-VT) with that of doxorubicin (DOX), a first-line chemotherapy drug, and tamoxifen (TAM), an endocrine therapy drug, on the proliferation of breast cancer cells. We found that Ad-VT could effectively inhibit the proliferation of breast cancer cells (p < 0.01); the inhibition rate of Ad-VT on normal mammary epithelial MCF-10A cells was less than 20%. DOX can effectively inhibit the proliferation of breast cancer cells and also has a strong inhibitory effect on MCF-10A cells (p < 0.01). TAM also has a strong inhibitory effect on breast cancer cells, among which the oestrogen-dependent MCF-7 cell inhibition was stronger (p < 0.01), At higher concentrations, TAM also had a high rate of inhibition (>70%) on the proliferation of MCF-10A cells. We also found that both recombinant adenovirus and both drugs could successfully induce tumour cell apoptosis. Further Western blot results showed that the recombinant adenovirus killed breast cancer cells through the endogenous apoptotic pathway. Analysis of the nude mouse subcutaneous breast cancer model showed that Ad-VT significantly inhibited tumour growth (the luminescence rate of cancer cells was reduced by more than 90%) and improved the survival rate of tumour-bearing mice (p < 0.01). Compared with DOX and TAM, Ad-VT has a significant inhibitory effect on breast cancer cells, but almost no inhibitory effect on normal breast epithelial cells, and this inhibitory effect is mainly through the endogenous apoptotic pathway. These results indicate that Ad-VT has significant potential as a drug for the treatment of breast cancer.

Myricetin activates the Caspase-3/GSDME pathway via ER stress induction of pyroptosis in lung cancer cells.

Pyroptosis is related to the occurrence, development, and therapeutic response of tumors, mediated by the proteins of the Gasdermin family. These proteins have become potential biomarkers for cancer treatment, and their agonists are likely to become a new direction in research and development of antitumor drugs. In this study, we found that myricetin has an inhibitory effect on lung cancer cells of the activation of pyroptosis. Analysis of the expression of Gasdermin family proteins revealed that this phenomenon was caused by the cleavage of GSDME. Subsequently, specific inhibitors, we found that caspase-3 was its upstream activation factor. In addition, mitochondrial and endoplasmic reticulum (ER) analysis showed that myricetin can cause endoplasmic reticulum stress and increase reactive oxygen species (ROS) levels. Subsequent inhibition of caspase-12 revealed that the expression levels of cleaved-caspase-3 and cleaved-GSDME were significantly reduced, resulting in the inhibition of pyroptosis. Using in vivo experiments, we also found that the treatment with myricetin can reduce tumor volume and significantly increase the level of pyroptosis-related proteins in tumor tissues. Overall, our findings show that myricetin induces cell death of lung cancer cells primarily through an ER stress pathway-induced pyroptosis. Therefore, myricetin has the potential to be used as a pyroptosis agonist in research and development of antitumor drugs.

Recombinant Oncolytic Adenovirus Combined with Cyclophosphamide Induces Synergy in the Treatment of Breast Cancer in vitro and in vivo.

Purpose: Oncolytic virus therapy has gradually become an integral approach in cancer treatment. We explored the therapeutic effects of the combination of a dual cancer-selective anti-tumor recombinant adenovirus (Ad-Apoptin-hTERTp-E1a) and cyclophosphamide on breast cancer cells. Methods: The inhibition of MCF-7 and MDA-MB-231 breast cancer cells by Ad-Apoptin-hTERTp-E1a (Ad-VT), cyclophosphamide, and Ad-VT + Cyclophosphamide was investigated using the CCK-8 assay. The combination index (CI) was calculated using CalcuSyn software to determine the best combination based on the inhibition rates of the different treatment combinations. The CCK-8 assay and crystal violet staining were used to detect the cytotoxicity of the combined Ad-VT and cyclophosphamide in breast cancer cells and breast epithelial cells. Subsequently, Hoechst staining, annexin V flow cytometry, and JC-1 staining were used to analyze the inhibitory pathway of Ad-VT plus cyclophosphamide on breast cancer cells. Cell migration and invasion of breast cancer cells were assessed using the cell-scratch and Transwell assays. The anti-tumor effects of different treatment groups in a tumor-bearing nude mouse model also were analyzed. Results: The treatment combination of Ad-VT (40 MOI) and cyclophosphamide (400 µM) significantly inhibited MCF-7 and MDA-MB-231 cells and reduced the toxicity of cyclophosphamide in normal cells. Ad-VT primarily induced breast cancer cell apoptosis through the endogenous apoptotic pathway. Apoptosis was significantly increased after treatment with Ad-VT plus cyclophosphamide. The combination significantly inhibited the migration and invasion of MCF-7 and MDA-MB-231 cells. The in vivo experiments demonstrated that exposure to Ad-VT plus cyclophosphamide significantly inhibited tumor growth and extended the survival time of the nude mice. Conclusion: Ad-VT plus cyclophosphamide reduced toxicity and exhibited increased efficacy in treating breast cancer cells.