A population study comparing tracheal and lung adenoid cystic carcinoma.

BACKGROUND: Thoracic adenoid cystic carcinoma (ACC) is rare, and the differences between tracheal and lung lesions have not been fully understood. METHODS: Patients were identified from a Chinese cancer center (FUSCC) (2005-2022) and the Surveillance, Epidemiology, and End Results (SEER) database (2000-2019). Incidence was calculated and trends were quantified. Clinicopathological features and overall survival (OS) were analyzed. Nomograms predicting OS were constructed. RESULTS: Totally, 55 tracheal adenoid cystic carcinoma (TACC) and 25 lung and bronchus adenoid cystic carcinoma (LACC) were included in a Chinese cohort, 121 TACC and 162 LACC included in the SEER cohort. There were larger tumor sizes, more lymph nodes and distant metastases for LACC than TACC patients. TACC patients are more likely to get local treatments. Patients with LACC had significantly worse median OS than patients with TACC (SEER cohort: 68.0 months vs. 109.0 months, p = 0.001, Chinese cohort: 62.9 months vs. 124.8 months, p = 0.061). Age, lymph node metastasis, distant metastasis and local treatment were identified as independent prognostic factors for OS of TACC. Distant metastasis and local treatment were identified for LACC. Specifically, surgery alone or in combination with radiotherapy is crucial for improving survival in both TACC and LACC. Only TACC benefits from radiotherapy alone, while chemotherapy does not improve survival for either. The nomograms constructed using these factors revealed good prognostic accuracy. CONCLUSIONS: LACC is more aggressive and has a worse prognosis than TACC. TACC patients have more opportunities for local treatment, which is important for the prognosis of both TACC and LACC. Nomograms were created for TACC and LACC to aid in personalized survival predictions and clinical decisions.

Systemic therapy with or without local intervention for oligometastatic oesophageal squamous cell carcinoma (ESO-Shanghai 13): an open-label, randomised, phase 2 trial.

BACKGROUND: The efficacy of local therapy for patients with oligometastatic oesophageal squamous cell carcinoma is unclear. We aimed to assess the efficacy of local plus systemic therapy compared with systemic therapy alone in patients with oligometastatic oesophageal squamous cell carcinoma. METHODS: The ESO-Shanghai 13 trial was a randomised, open-label, multicentre, phase 2 trial. Patients (aged ≥18 years) were recruited from six hospitals in China with histological confirmation of oligometastatic oesophageal squamous cell carcinoma with a controlled primary tumour and one to four metastatic lesions. Eligible patients were randomly assigned via a computer-generated schedule in a 1:1 ratio to receive either systemic therapy alone (ie, systemic therapy only group) or combined systemic and local therapy (ie, systemic and local therapy group). The systemic therapy regimens in both groups were at the discretion of the investigator and included chemotherapy alone, anti-PD-1 antibodies alone, or chemotherapy plus anti-PD-1 antibodies. Local therapy-radiotherapy, surgery, or thermal ablation-was delivered to all metastatic lesions for patients in the systemic and local therapy group. Randomisation was balanced dynamically on three factors: the number of disease sites, the lines of systemic therapy, and the location of the metastases. Patients and investigators were not masked to treatment allocation. The primary endpoint was progression-free survival, defined as the time from randomisation to progression or death from any cause in the intention-to-treat population. The safety population included all patients who had undergone random assignment and at least one of the intended therapies. This trial is registered with ClinicalTrials.gov, NCT03904927. The trial is ongoing but closed to new participants. FINDINGS: 116 patients were screened for enrolment between March 5, 2019, and Sept 16, 2021, and 104 patients who met the eligibility criteria were randomly assigned to the systemic and local therapy group (n=53) or the systemic therapy only group (n=51). 20 (38%) patients in the systemic plus local therapy group and 23 (45%) patients in the systemic therapy only group received anti-PD-1 antibody-based systemic therapy; three patients in the systemic and local therapy group did not receive systemic therapy. At a median follow-up of 30·5 months (IQR 24·7-37·8), median progression-free survival was 15·3 months (95% CI 10·1-20·5) in the systemic and local therapy group versus 6·4 months (5·2-7·6) in the systemic therapy only group (stratified hazard ratio 0·26 [95% CI 0·16-0·42]; stratified log rank p<0·0001). Grade 1-2 acute oesophagitis was more common in the systemic and local therapy group than in the systemic therapy only group (10 [19%] vs one [2%] patients; p=0·036). The number of patients who had grade 3 or worse treatment-related adverse events was similar between groups (25 [47%] vs 21 [41%]; p=0·538), with the most common adverse events being leukocytopenia (17 [32%] vs 18 [35%]) and neutropenia (19 [36%] vs 20 [39%]). Treatment-related deaths occurred in two patients in the systemic and local therapy group and one patient in the systemic therapy only group. INTERPRETATION: The addition of local treatment for metastases could significantly improve progression-free survival among patients with oligometastatic oesophageal squamous cell carcinoma being treated with systemic therapy. Our findings suggest that combining local and systemic therapy could be a treatment option for patients with oligometastatic oesophageal squamous cell carcinoma, but further support from phase 3 trials is required. FUNDING: Science and Technology Commission of Shanghai Municipality, National Nature Science Foundation of China, and Shanghai Municipal Health Commission. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

Correction to: Targeted therapy for cisplatin-resistant lung cancer via aptamer-guided nano-zinc carriers containing USP14 siRNA.

[This corrects the article DOI: 10.1002/mco2.237.].

Targeted therapy for cisplatin-resistant lung cancer via aptamer-guided nano-zinc carriers containing USP14 siRNA.

Cisplatin (DDP) is a common therapeutic option for non-small cell lung carcinoma (NSCLC). However, some patients fail to respond to the DDP chemotherapy. Therefore, identifying novel biomarkers to improve the diagnosis and treatment of NSCLC is important. Ubiquitin-specific protease (USP14) is involved in various pathological conditions including cancer; however, the role of USP14 in NSCLC remains elusive. The SELEX technology was used to identify aptamers that specifically recognize DDP-resistant lung cancer cells and couple them with nano-zinc (zinc hydroxide, Zn(OH)2) carriers. USP14 levels were higher in DDP-resistant lung cancer compared to DDP-sensitive lung cancer. The survival rate of lung cancer patients with increased USP14 expression was significantly lower than the survival rate of patients with low USP14 expression. Silencing USP14 increased the tumor antagonistic action of DDP in A549 cisplatin-resistant (A549/DDP) cells, while USP14 overexpression decreased the antagonist effects. Aptamer-targeted nano-zinc carriers were loaded with USP14 siRNA to target DDP-resistant lung cancer cells. Aptamer-targeted nano-zinc carriers containing USP14 siRNA increased the antitumor effects of DDP in A549/DDP cells and mice bearing A549/DDP cells. These results indicate that aptamer-guided nano-zinc carriers may be a potent carrier for the precise treatment of drug-resistant tumors.

Comparison of treatment strategies for resectable locally advanced primary mucinous adenocarcinoma of the lung.

BACKGROUND: Primary pure mucinous adenocarcinoma (PMA) is a rare type of lung cancer with unique clinical and prognostic features. Previous studies have shown that PMA have more early-stage cancer compared with other adenocarcinoma (ADC) subtypes. The clinicopathological features and optimal treatment strategies of resectable locally advanced mucinous adenocarcinoma lack evidence and require further study. METHODS: In this study, we collected information from patients with stage III-N2 PMA who underwent radical surgery between 2004 and 2016 from the Surveillance, Epidemiology, and End Results (SEER) database. The clinicopathological parameters, treatments, overall survival (OS), and cancer-specific survival (CSS) were evaluated. RESULTS: Of 242,699 eligible lung adenocarcinoma patients, 124 with PMA and 3405 with other ADCs of stage III-N2 received radical surgery were identified. Compared with other ADCs, PMA tended to appear more in the lower lobes, with higher degree of differentiation, less early T stage, and more positive lymph nodes numbers. Patients with PMA had significantly worse survival than other ADCs (OS = 45.0 vs. 57.1 months, p = 0.005, CSS = 51.8 vs. 65.5 months, p = 0.017). We explored the benefit population of postoperative radiotherapy (PORT) and found that the population with ≤7 positive lymph nodes could benefit from PORT, and OS was significantly improved (41.2 vs. 69.3 months, p = 0.034). For patients with >7 positive lymph nodes, PORT did not provide a survival benefit, while chemotherapy improved OS (10.9 vs. 23.3 months, p = 0.041). Multivariate analysis showed that race, tumor location, number of positive lymph nodes, and PORT were independent prognostic factors in patients with postoperative III-N2 lung PMA. CONCLUSION: The prognosis of patients with resectable III-N2 primary lung PMA was significantly worse than that of other ADCs, and PORT was an independent prognostic factor. Patients with ≤7 positive lymph nodes could benefit from PORT and those with >7 positive lymph nodes could benefit from chemotherapy.

In Vivo 3-D Dose Verification Using PET/CT Images After Carbon-Ion Radiation Therapy.

OBJECTIVE: To investigate the usefulness of positron emission tomography (PET) images obtained after carbon-ion irradiation for dose verification in carbon-ion radiotherapy. METHODS AND MATERIALS: An anthropomorphic head phantom was used in this study. Three cubes with volumes of 1, 4, and 10 ml were contoured as targets in the phantom CT through a treatment planning system. Treatment plans with six prescriptions from 2.5 to 10 Gy (2.5, 3, 5, 6, 8, and 10 Gy effective dose) were designed and delivered by 90° fixed carbon-ion beams, respectively. After irradiation of the phantom, a PET/CT scan was performed to fuse the treatment-planning CT image with the PET/CT image. The relationship between target volume and the standard uptake value (SUV) in PET/CT was evaluated for corresponding plan prescription. The MIM Maestro software was used for the image fusion and data analysis. RESULTS: SUV in the target had an approximate linear relationship with the effective dose. The same effective dose could generate a roughly equal SUV for different target volumes (p < 0.05). CONCLUSIONS: It is feasible to verify the actual 3-D dose distribution of carbon-ion radiotherapy by the approach in this study.

Local therapy for oligometastatic esophageal squamous cell carcinoma: a prospective, randomized, Phase II clinical trial.

For patients with oligometastatic esophageal squamous cell carcinoma, the efficacy of local therapy is still controversial because of patient selection and lack of adequate controls in most studies. Here the authors design the ESO-Shanghai 13 trial, a prospective, multicenter, randomized, Phase II trial, to assess the impact of combined local therapy and systemic therapy on progression and survival compared with systemic therapy alone for patients with four or less metastases. A total of 102 patients will be recruited over 3 years from approximately five centers and randomized in a 1:1 ratio to receive either systemic therapy alone or systemic therapy and local therapy, such as radiation, surgery and thermal ablation. The primary endpoint is progression-free survival. The secondary endpoints are overall survival, local control, toxicity and quality of life. Clinical trial registration: NCT03904927 (ClinicalTrials.gov).

Lay abstract The ESO-Shanghai 13 trial is a prospective, multicenter, randomized, Phase II trial to assess the impact of combined local treatment (such as radiotherapy, surgery and thermal ablation) and chemical drugs for patients with esophageal squamous cell carcinoma. Patients with four or less metastases and controlled esophageal lesion will be enrolled. The authors will recruit a total of 102 patients over 3 years from approximately five centers. All patients will be randomized and receive either chemical drugs alone or chemical drugs plus local treatment with the same probability. Patients will then be observed after treatment until disease progression or death or the end of the trial. Patients will need to report their symptoms and physical status and fill out quality of life scales during the treatment and follow-up period.

AZD9291 Resistance Reversal Activity of a pH-Sensitive Nanocarrier Dual-Loaded with Chloroquine and FGFR1 Inhibitor in NSCLC.

AZD9291 can effectively prolong survival of non-small cell lung cancer (NSCLC) patients. Unfortunately, the mechanism of its acquired drug resistance is largely unknown. This study shows that autophagy and fibroblast growth factor receptor 1 signaling pathways are both activated in AZD9291 resistant NSCLC, and inhibition of them, respectively, by chloroquine (CQ) and PD173074 can synergistically reverse AZD9291 resistance. Herein, a coloaded CQ and PD173074 pH-sensitive shell-core nanoparticles CP@NP-cRGD is developed to reverse AZD9291 resistance in NSCLC. CP@NP-cRGD has a high encapsulation rate and stability, and can effectively prevent the degradation of drugs in circulation process. CP@NP-cRGD can target tumor cells by enhanced permeability and retention effect and the cRGD peptide. The pH-sensitive CaP shell can realize lysosome escape and then release drugs successively. The combination of CP@NP-cRGD and AZD9291 significantly induces a higher rate of apoptosis, more G0/G1 phase arrest, and reduces proliferation of resistant cell lines by downregulation of p-ERK1/2 in vitro. CQ in CP@NP-cRGD can block protective autophagy induced by both AZD9291 and PD173074. CP@NP-cRGD combined with AZD9291 shows adequate tumor enrichment, low toxicity, and excellent antitumor effect in nude mice. It provides a novel multifunctional nanoparticle to overcome AZD9291 resistance for potential clinical applications.

Lactate-Modulated Immunosuppression of Myeloid-Derived Suppressor Cells Contributes to the Radioresistance of Pancreatic Cancer.

The mechanisms responsible for radioresistance in pancreatic cancer have yet to be elucidated, and the suppressive tumor immune microenvironment must be considered. We investigated whether the radiotherapy-augmented Warburg effect helped myeloid cells acquire an immunosuppressive phenotype, resulting in limited treatment efficacy of pancreatic ductal adenocarcinoma (PDAC). Radiotherapy enhanced the tumor-promoting activity of myeloid-derived suppressor cells (MDSC) in pancreatic cancer. Sustained increase in lactate secretion, resulting from the radiation-augmented Warburg effect, was responsible for the enhanced immunosuppressive phenotype of MDSCs after radiotherapy. Hypoxia-inducible factor-1α (HIF-1α) was essential for tumor cell metabolism and lactate-regulated activation of MDSCs via the G protein-coupled receptor 81 (GPR81)/mTOR/HIF-1α/STAT3 pathway. Blocking lactate production in tumor cells or deleting Hif-1α in MDSCs reverted antitumor T-cell responses and effectively inhibited tumor progression after radiotherapy in pancreatic cancer. Our investigation highlighted the importance of radiation-induced lactate in regulating the inhibitory immune microenvironment of PDAC. Targeting lactate derived from tumor cells and the HIF-1α signaling in MDSCs may hold distinct promise for clinical therapies to alleviate radioresistance in PDAC.

Circulating neutrophil-to-lymphocyte ratio at admission predicts the long-term outcome in acute traumatic cervical spinal cord injury patients.

BACKGROUND: The prognostic value of Neutrophil-to-Lymphocyte Ratio (NLR) for the outcome of acute cervical traumatic spinal cord injury (tSCI) patients has rarely been studied by now throughout the world. METHODS: We performed a single-center retrospective cohort study to evaluate the prognostic value of NLR from peripheral whole blood count in patients with acute cervical tSCI. Patients within 6 h of acute cervical tSCI treated between Dec 2008 and May 2018 in Huashan Hospital of Fudan University were enrolled. Outcomes of patients with tSCI were assessed using American spinal injury association Impairment Scale (AIS). 6-month outcomes were dichotomized into poor outcome group (AIS A to C) and good outcome group (AIS D and E). Uni- and multivariate analyses were performed to assess the independent predictors of 6-month outcome. Two prediction models based on admission characteristics were built to evaluate the prognostic value of NLR. The discriminative ability of predictive models was evaluated using the area under the curve (AUC). RESULTS: A total of 377 patients were identified from our single center in China PR. Multivariate analysis showed that age, AIS grade at admission, NLR (p < 0.001) and coagulopathy (p = 0.003) were independent predictors of the 6-months outcome for acute cervical tSCI patients. The model combing NLR and standard variables (AUC = 0.944; 95% CI, 0.923-0.964) showed a more favorable prognostic value than that without NLR (AUC = 0.841; 95% CI, 0.798-0.885) in terms of 6-month outcome. CONCLUSIONS: NLR is firstly identified as an independent predictor of the 6-month outcome in acute cervical tSCI patients worldwide. The prognostic value of NLR is favorable, and a high NLR is associated with poor outcome in patients with acute cervical tSCI.

Astragaloside IV sensitizes non-small cell lung cancer cells to cisplatin by suppressing endoplasmic reticulum stress and autophagy.

BACKGROUND: Cisplatin is an effective chemotherapeutic drug for treating various cancers including non-small cell lung cancer (NSCLC), but resistance to cisplatin remains the main limitation to its use in clinic. Astragaloside IV (AS-IV), which is derived from Astragalus membranaceus, has been proven to participate in various anti-cancer activities including anti-cancer, anti-oxidative, and anti-inflammatory functions. METHOD: In this study, we explored the role of AS-IV in cisplatin chemoresistance to NSCLC cells by establishing cisplatin-resistant the NSCLC cell lines, A549Cis and H1299Cis. RESULTS: Cisplatin inhibited viability and promoted apoptosis of A549Cis and H1299Cis cells in a dose-dependent manner. In addition, cisplatin upregulated the levels of autophagy-related proteins (Beclin1, LC3 II/I) and endoplasmic reticulum (ER) stress-related proteins (glucose regulated protein 78: GRP78, protein kinase R (PKR)-like endoplasmic reticulum kinase: PERK), indicating that cisplatin caused autophagy and ER stress in NSCLC cells. However, treatment combined with AS-IV dose-dependently suppressed cell viability and increased the cell apoptosis rate in A549Cis and H1299Cis cells, suggesting that AS-IV elevated the anti-tumor role of cisplatin in NSCLC cells. AS-IV treatment suppressed the expression of GRP78 and Beclin1. Inhibition of ER stress or autophagy both counteracted the inhibitory effect of AS-IV on chemoresistance to cisplatin in NSCLC cells. CONCLUSIONS: AS-IV sensitized NSCLC cells to cisplatin through suppressing ER stress and autophagy. This study provides a novel strategy of cisplatin combined with AS-IV for the treatment of cisplatin-resistant NSCLC patients.

Radiosensitization of metformin in pancreatic cancer cells via abrogating the G2 checkpoint and inhibiting DNA damage repair.

Recent evidences have demonstrated the potential of metformin as a novel agent for cancer prevention and treatment. Here, we investigated its ability of radiosensitization and the underlying mechanisms in human pancreatic cancer cells. In this study, we found that metformin at 5 mM concentration enhanced the radiosensitivity of MIA PaCa-2 and PANC-1 cells, with sensitization enhancement ratios of 1.39 and 1.27, respectively. Mechanistically, metformin caused abrogation of the G2 checkpoint and increase of mitotic catastrophe, associated with suppression of Wee1 kinase and in turn CDK1 Tyr15 phosphorylation. Furthermore, metformin inhibited both expression and irradiation-induced foci formation of Rad51, a key player in homologous recombination repair, ultimately leading to persistent DNA damage, as reflected by γ-H2AX and 53BP1 signaling. Finally, metformin-mediated AMPK/mTOR/p70S6K was identified as a possible upstream pathway controlling translational regulation of Wee1 and Rad51. Our data suggest that metformin radiosensitizes pancreatic cancer cells in vitro via abrogation of the G2 checkpoint and inhibition of DNA damage repair. However, the in vivo study is needed to further confirm the findings from the in vitro study.

Metformin is associated with reduced risk of pancreatic cancer in patients with type 2 diabetes mellitus: a systematic review and meta-analysis.

AIMS: Recent epidemiological studies indicated that use of metformin might decrease the risk of various cancers among patients with type 2 diabetes mellitus (T2DM). However, its influence on pancreatic cancer was controversial. Therefore, we did a meta-analysis of currently available observational studies on the issue. METHODS: We did a PubMed and ISI Web of Science search for observational articles. The pooled relative risk (RR) was estimated using a random-effect model. Heterogeneity was evaluated using I(2) statistic. Subgroup analysis was performed to explore the source of heterogeneity and confirm the overall estimates. Publication bias was also examined. RESULTS: The analysis included 11 articles (13 studies) comprising 10 cohort studies and 3 case-control studies. Use of metformin was associated with a significant lower risk of pancreatic cancer [RR 0.63, 95% confidence internal (CI) 0.46-0.86, p=0.003]. In a total 11 subgroup analyses, 5 provided the consistent result with pooled effect estimates of overall analysis. No publication bias was detected by Begg's (Z=-0.79, p=0.428) and Egger's test (t=-0.92, p=0.378). CONCLUSIONS: From present observational studies, use of metformin appears to be associated with a reduced risk of pancreatic cancer in patients with T2DM. Further investigation is needed.

Safety and efficacy of nimotuzumab in combination with radiotherapy for patients with squamous cell carcinoma of the esophagus.

BACKGROUND: We investigated nimotuzumab (h-R3), a humanized monoclonal antibody against epidermal growth factor receptor, when combined with irradiation or chemoradiation for squamous cell carcinoma (SCC) of the esophagus. The aim of this study was to evaluate its safety and efficacy. METHODS: We retrospectively analyzed 66 patients with esophageal SCC treated with a combination of h-R3 and radiation or chemoradiation between December 2008 and September 2011 at Fudan University Shanghai Cancer Center. Fifty-two of the 66 patients received h-R3 combined with chemoradiation and 14 received h-R3 plus radiation. The median total irradiation dose was 61 Gy given by conventional fractionation. The h-R3 weekly dosage was 100 mg (6/66), 200 mg (54/66), or 400 mg (6/66) given concurrently during the irradiation period. RESULTS: Patients tolerated the treatment well. Grade 3-4 adverse events and toxicities occurred in 50 % of the patients. h-R3-related toxicities manifested as Grade 1 skin rash in 1 case and Grade 2 infusion-related reaction in 2 cases. The median overall survival (OS) and progression-free survival (PFS) were 26.0 months and 16.7 months, respectively. OS, PFS and locoregional control (LC) at 2 years were 54, 37 and 80 %, respectively. CONCLUSIONS: h-R3 in combination with irradiation or chemoradiation was safe and tolerable, and yielded encouraging OS, PFS and LC.

Sorafenib potentiates irradiation effect in hepatocellular carcinoma in vitro and in vivo.

The multikinase-inhibition action of sorafenib provides strong rationales for its combination use with radiotherapy. We investigated the in vitro and in vivo effect of sorafenib combined with irradiation on hepatocellular carcinoma (HCC). Sorafenib enhanced radiosensitivity of human HCC cell lines in a schedule-dependent manner. Sorafenib selectively inhibited radiation-induced activation of vascular endothelial growth factor receptor-2 (VEGFR2) and downstream extracellular signal-regulated kinase (ERK) pathway, induced DNA damage and suppressed DNA repair capacity, decreased radiation-activated NF-κB and increased radiation-induced apoptosis. In xenograft experiments, combination treatment produced marked tumor growth delay in both concurrent and sequential schedules. These results suggest that sorafenib could potentiate irradiation effect in HCC, which warrants further investigation for its potential clinical applications.

Hepatic regeneration after sublethal partial liver irradiation in cirrhotic rats.

Our previous animal study had demonstrated that partial liver irradiation (IR) could stimulate regeneration in the protected liver, which supported the measurements adopted in radiotherapy planning for hepatocellular carcinoma. The purpose of this present study is to investigate whether cirrhotic liver repopulation could be triggered by partial liver IR. The cirrhosis was induced by thioacetamide (TAA) in rats. After cirrhosis establishment, TAA was withdrawn. In Experiment 1, only right-half liver was irradiated with single doses of 5 Gy, 10 Gy and 15 Gy, respectively. In Experiment 2, right-half liver was irradiated to 15 Gy, and the left-half to 2.5 Gy, 5 Gy and 7.5 Gy, respectively. The regeneration endpoints, including liver index (LI); mitotic index (MI); liver proliferation index (LPI); PCNA-labeling index (PCNA-LI); serum HGF, VEGF, TGF-α and IL-6, were evaluated on 0 day, 30-day, 60-day, 90-day, 120-day and 150-day after IR. Serum and in situ TGF-ß1 were also measured. In both experimental groups, the IR injuries were sublethal, inducing no more than 9% animal deaths. Upon TAA withdrawal, hepatic regeneration decelerated in the controls. In Experiment 1 except for LI, all other regeneration parameters were significantly higher than those in controls for both right-half and left-half livers. In Experiment 2 all regeneration parameters were also higher compared with those in controls for both half livers. Serum HGF and VEGF were increased compared with that of controls. Both unirradiated and low dose-irradiated cirrhotic liver were able to regenerate triggered by sublethal partial liver IR and higher doses and IR to both halves liver triggered a more enhanced regeneration.

A natural process of cirrhosis resolution and deceleration of liver regeneration after thioacetamide withdrawal in a rat model.

Characteristics of thioacetamide (TAA)-induced liver cirrhosis in rat was observed for 120 days after TAA withdrawal as part of the radiobiological study of partial liver irradiation on TAA-induced cirrhotic rats. The natural process focused on cirrhosis and regeneration was recorded as a baseline condition for the interpretation of the outcome of the partial liver irradiation study. Cirrhosis in rats was successfully induced by drinking 0.03% TAA water orally for 29 weeks with a modeling rate of 96%. After establishment of the cirrhosis model, the rats were observed for 120 days upon TAA withdrawal to investigate the dynamic changes of cirrhosis and regeneration. The following characteristics were observed: (1) Histological changes; (2) Liver functions; (3) Cirrhosis: trichrome stain, quantification of hydroxyproline in hydrolysed liver tissue and TGF-ß1; (4) Liver regeneration: liver index, hepatocyte mitotic index (MI), hepatocyte proliferation index (PI) by flow cytometry, PCNA labeling index (LI) by IHC and expression of PCNA mRNA; and (5) Growth factors: serum HGF, VEGF, TGF-α, and IL-6. After TAA withdrawal, gradual improvement in liver functions was noted with decreases of ALT, AST, and ALP, and increase of PA. The resolution of cirrhosis was evident by histological improvement with attenuation of collagen fiber and decrease of TGF-ß1 IHC index, and also decrease of trichrome stain and hydroxyproline content. However, cirrhosis was still existed on 120 days after TAA withdrawal. Significant deceleration of liver regeneration was demonstrated with TAA withdrawal, evidenced by decrease of MI and PI, reduced expression of PCNA mRNA and PCNA LI. In conclusion, upon TAA withdrawal hepatic cirrhosis was continuously resolved, but persisted up to 120 days, and liver regeneration was significantly decelerated.

Improved therapeutic efficacy against murine carcinoma by combining honokiol with gene therapy of PNAS-4, a novel pro-apoptotic gene.

PNAS-4, a novel pro-apoptotic gene activated during the early response to DNA damage, can inhibit proliferation via apoptosis when overexpressed in some tumor cells. Recent studies have indicated that honokiol can induce apoptosis, inhibit angiogenesis, and suppress tumor growth. In the present study, we investigated whether mouse PNAS-4 (mPNAS-4) could augment the apoptosis of tumor cells induced by honokiol in vitro, and whether the antiangiogenic activity of honokiol and induction of apoptosis by mPNAS-4 could work cooperatively to improve the antitumor efficacy in vivo. In vitro, mPNAS-4 inhibited proliferation of murine colorectal carcinoma CT26 and Lewis lung carcinoma LL2 cells through induction of apoptosis, and significantly augmented the apoptosis of CT26 and LL2 cells induced by honokiol. Compared with treatment with mPNAS-4 or honokiol alone, in vivo systemic administration of an expression plasmid encoding mPNAS-4 and low-dose honokiol significantly suppressed tumor growth through the enhanced induction of apoptosis and the augmented inhibition of angiogenesis. Our data suggest that the combined treatment with mPNAS-4 plus honokiol augments antitumor effects in vitro and in vivo, and that the improved antitumor activity in vivo may be associated with enhanced induction of apoptosis and augmented inhibition of angiogenesis. The present study may provide a novel and effective method for the treatment of cancer.

PNAS-4, a novel pro-apoptotic gene, can potentiate antineoplastic effects of cisplatin.

PURPOSE: PNAS-4, a novel pro-apoptotic gene activated during the early response to DNA damage, can inhibit proliferation via apoptosis when overexpressed in some tumor cells. The objectives of this study were to determine whether PNAS-4 could enhance apoptosis induced by cisplatin besides its induction of apoptosis, and to evaluate the usefulness of combined treatment with mouse PNAS-4 (mPNAS-4) gene therapy and low-dose cisplatin chemotherapy in the inhibition of tumor growth in colon carcinoma (CT26) and Lewis lung carcinoma (LL/2) murine models. METHODS: In this study, the in vitro growth-inhibitory and pro-apoptotic effects of PNAS-4 and/or cisplatin on CT26, LL/2, and SKOV3 cancer cells were assessed by MTT assay, flow cytometric analysis, DNA fragmentation, and morphological analysis, respectively. The in vivo antitumor activity of combined treatment with mPNAS-4 gene therapy and low-dose cisplatin were evaluated in the inhibition of tumor growth in colon carcinoma (CT26) and Lewis lung carcinoma (LL/2) murine models. Tumor volume and survival time were observed. Induction of apoptosis was also assessed in tumor tissues. RESULTS: In vitro, PNAS-4 inhibited proliferation of colon carcinoma (CT26), Lewis lung carcinoma (LL/2) and human ovarian cancer (SKOV3) cell lines via apoptosis, and significantly enhanced the apoptosis of CT26, LL/2, and SKOV3 cells induced by cisplatin. In vivo systemic administration of expression plasmid encoding mPNAS-4 (pcDNA3.1-mPS) and cisplatin, significantly decreased tumor growth through increased tumor cell apoptosis compared to treatment with mPNAS-4 or cisplatin alone. CONCLUSIONS: Our data suggests that the combined treatment with mPNAS-4 plus cisplatin may augment the induction of apoptosis in tumor cells in vitro and in vivo, and that the augmented antitumor activity in vivo may result from the increased induction of apoptosis. The present study may provide a novel way to augment the antitumor efficacy of cytotoxic chemotherapy.

A novel pro-apoptosis protein PNAS-4 from Xenopus laevis: cloning, expression, purification, and polyclonal antibody production.

Human PNAS-4 was identified as a novel pro-apoptotic protein in mammalian cells. Here we report the cloning, expression, purification, and antibody production of a PNAS-4 homolog (named xPNAS-4) from Xenopus laevis, an extensively used model organism in exploring gene functions during embryonic development. Recombinant histidine-tagged xPNAS-4 protein was expressed in Escherichia coli as insoluble inclusion bodies. The inclusion bodies were subsequently dissolved in 8 M urea and purified to near homogeneity by Ni2+ affinity chromatography. The resulting denatured protein was refolded by stepwise dilution of urea concentration via dialysis. This procedure yielded about 4 mg refolded protein per liter of E. coli culture with a purity of 95%. The purified protein was identified by liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (LC-ESI-Q-TOF-MS) and used to raise anti-xPNAS-4 polyclonal antibodies that were suitable for detecting the expression of PNAS-4 in X. laevis embryos by Western blotting. The availability of recombinant protein and specific polyclonal antibodies will provide a valuable tool in studying apoptotic mechanisms of this protein. To our knowledge, this is the first report to demonstrate the presence of PNAS-4 in X. laevis.