Laboratory scoring system to predict hepatic indocyanine green clearance ability during fluorescence imaging-guided laparoscopic hepatectomy.

BACKGROUND: Indocyanine green (ICG) fluorescence played an important role in tumor localization and margin delineation in hepatobiliary surgery. However, the preoperative regimen of ICG administration was still controversial. Factors associated with tumor fluorescence staining effect were unclear. AIM: To investigate the preoperative laboratory indexes corelated with ICG fluorescence staining effect and establish a novel laboratory scoring system to screen specifical patients who need ICG dose adjustment. METHODS: To investigate the predictive indicators of ICG fluorescence characteristics in patients undergoing laparoscopic hepatectomy from January 2018 to January 2021 were included. Blood laboratory tests were completed within 1 wk before surgery. All patients received 5 mg ICG injection 24 h before surgery for preliminary tumor imaging. ImageJ software was used to measure the fluorescence intensity values of regions of interest. Correlation analysis was used to identify risk factors. A laboratory risk model was established to identify individuals at high risk for high liver background fluorescence. RESULTS: There were 110 patients who were enrolled in this study from January 2019 to January 2021. The mean values of fluorescence intensity of liver background (FI-LB), fluorescence intensity of gallbladder, and fluorescence intensity of target area were 18.87 ± 17.06, 54.84 ± 33.29, and 68.56 ± 36.11, respectively. The receiver operating characteristic (ROC) curve showed that FI-LB was a good indicator for liver clearance ability [area under the ROC curve (AUC) = 0.984]. Correlation analysis found pre-operative aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transpeptidase, adenosine deaminase, and lactate dehydrogenase were positively associated with FI-LB and red blood cell, cholinesterase, and were negatively associated with FI-LB. Total laboratory risk score (TLRS) was calculated according to ROC curve (AUC = 0.848, sensitivity = 0.773, specificity = 0.885). When TLRS was greater than 6.5, the liver clearance ability of ICG was considered as poor. CONCLUSION: Preoperative laboratory blood indicators can predict hepatic ICG clearance ability. Surgeons can adjust the dose and timing of ICG preoperatively to achieve better liver fluorescent staining.

Complete Response to Combined Chemotherapy and Anti-PD-1 Therapy for Recurrent Gallbladder Carcinosarcoma: A Case Report and Literature Review.

Background: Gallbladder carcinosarcoma (GBCS) is a rare and aggressive malignancy with extremely poor prognosis. Although surgery is regarded as the primary therapy for GBCS, the effective therapeutic strategies for unresected lesions have been poorly defined. Case Presentation: We presented a case of a 74-year-old male who underwent radical resection of gallbladder carcinoma at a local hospital. Seven months later, he was admitted to our hospital due to right upper abdominal discomfort. Postoperative radiological examinations showed multiple hepatic lesions, hilar lymph node metastasis, and main portal vein tumor thrombus. The pathological consultation results confirmed GBCS and immunohistochemical examinations revealed PD-L1 expression in 20% of tumor cells. Then, the patient received chemotherapy (Gemcitabine plus Oxaliplatin, GEMOX) in combination with anti-PD-1 therapy. After nine courses of the combination therapy, complete regression of the tumors was achieved with no evidence of relapse till now. Conclusions: We, for the first time, reported a patient with recurrent GBCS who benefited from the combined chemotherapy and immunotherapy, providing a potential effective management strategy for the refractory malignant tumor.

Functional anatomical hepatectomy guided by indocyanine green fluorescence imaging in patients with localized cholestasis: Report of four cases.

BACKGROUND: Liver cancer is a malignant tumor with a high incidence. At present, the most effective treatment is laparoscopic hepatectomy (LH). Indocyanine green fluorescence imaging (ICG-FI) has become an important tool in LH, and the most common fluorescent types of tumors are total fluorescence, partial fluorescence, and rim fluorescence. CASE SUMMARY: We presented four cases of LH guided by ICG-FI in which we also observed the fourth special fluorescent type. When the tumor or intrahepatic stone compresses the adjacent bile duct to cause local cholestasis, the liver segment or subsegment with obstructed bile drainage will show strong fluorescence. Complete removal of the lesion together with the fluorescent liver parenchyma may help reduce the risk of tumor or stone recurrence. CONCLUSION: This type of partial fluorescence can indicate local biliary compression, and the resection method is related to bile drainage, which may be called functional anatomical hepatectomy and ensures radical resection of the lesion.

Osteoblast-derived lipocalin-2 regulated by miRNA-96-5p/Foxo1 advances the progression of Alzheimer's disease.

Aim: Alzheimer's disease (AD) is the most frequent cause of dementia and characterized by the accumulation of ß-amyloid peptides in plaques and vessel walls. This study proposed a hypothesis of an inhibitory role of miR-96-5p in AD via regulating Foxo1. Methods & methods: AD mouse models were established by injecting with 1% pentobarbital. Results: Knockdown of miR-96-5p in the presence of naringin was shown to reduce the expression of Foxo1 and contents of superoxide dismutase, catalase and glutathione peroxidase, yet increase lipocalin-2 expression as well as hydroxyproline and malondialdehyde contents. Also, Foxo1-mediated lipocalin-2 inhibition attenuated AD. Conclusion: Our study shows downregulating miR-96-5p limited AD progression, highlighting miR-96-5p a potential therapeutic target in treating AD.

Shikonin and 4-hydroxytamoxifen synergistically inhibit the proliferation of breast cancer cells through activating apoptosis signaling pathway in vitro and in vivo.

BACKGROUND: Tamoxifen (TAM) is a cell type-specific anti-estrogen and is applied to improve the survival of patients with estrogen receptor positive (ER +) breast cancer. However, long-term TAM use can induce serious drug resistance, leading to breast cancer recurrence and death in patients. Further, it is almost useless among patients with estrogen receptor negative (ER -) breast cancer. Shikonin (SK) is a natural product broadly explored in cancer therapy. Some studies have demonstrated the combined treatment of SK and clinical anticancer drugs including TAM on various tumors. However, the combined effect of SK and 4-hydroxytamoxifen (4-OHT) on ER- breast cancer is not known. The current study aimed to assess the combination effects of SK and 4-OHT on human breast cancer cells, MCF-7 (ER +) and MDA-MB-435S (ER -), in vitro and in vivo and to investigate the underlying mechanisms. METHODS: CCK-8 assays and flow cytometry were conducted to determine the cell viability and apoptotic profiles of human breast cancer cell lines (MCF-7 and MDA-MB-435S) treated with SK, 4-OHT, and the combination. ROS and JC-1 assays were used to determine ROS level and mitochondrial membrane potential. Western blot analysis was performed to investigate proteins that are associated with apoptosis. Haematoxylin & Eosin (HE) staining was used to detect the tumor and kidney morphology of mice. TUNEL and immunohistochemical staining were performed to detect Ki67 expression level and cell apoptotic profile in tumor tissues. RESULTS: SK and 4-OHT synergistically inhibited MCF-7 and MDA-MB-435S cell proliferation and promoted apoptosis by reducing mitochondrial membrane potential and increasing the intracellular ROS level. The combination of SK and 4-OHT activated the mitochondrial-dependent apoptosis and the death receptor pathways, significantly regulating the PI3K/AKT/Caspase 9 signaling pathway. Compared with SK and 4-OHT alone, the combination of SK and 4-OHT could better inhibit tumor growth in mice. CONCLUSION: The combination of SK and 4-OHT shows highly efficient anticancer effects on breast cancer therapy. SK may be a promising candidate as an adjuvant to 4-OHT for breast cancer treatments, especially for ER- breast cancer.

Design, synthesis and biological evaluation of benzoylacrylic acid shikonin ester derivatives as irreversible dual inhibitors of tubulin and EGFR.

In this study, a series of shikonin derivatives combined with benzoylacrylic had been designed and synthesized, which showed an inhibitory effect on both tubulin and the epidermal growth factor receptor (EGFR). In vitro EGFR and cell growth inhibition assay demonstrated that compound PMMB-317 exhibited the most potent anti-EGFR (IC50 = 22.7 nM) and anti-proliferation activity (IC50 = 4.37 µM) against A549 cell line, which was comparable to that of Afatinib (EGFR, IC50 = 15.4 nM; A549, IC50 = 6.32 µM). Our results on mechanism research suggested that, PMMB-317 could induce the apoptosis of A549 cells in a dose- and time-dependent manner, along with decrease in mitochondrial membrane potential (MMP), production of ROS and alterations in apoptosis-related protein levels. Also, PMMB-317 could arrest cell cycle at G2/M phase to induce cell apoptosis, and inhibit the EGFR activity through blocking the signal transduction downstream of the mitogen-activated protein MAPK pathway and the anti-apoptotic kinase AKT pathway; typically, such results were comparable to those of afatinib. In addition, PMMB-317 could suppress A549 cell migration through the Wnt/ß-catenin signaling pathway in a dose-dependent manner. Additionally, molecular docking simulation revealed that, PMMB-317 could simultaneously combine with EGFR protein (5HG8) and tubulin (1SA0) through various forces. Moreover, 3D-QSAR study was also carried out, which could optimize our compound through the structure-activity relationship analysis. Furthermore, the in vitro and in vivo results had collectively confirmed that PMMB-317 might serve as a promising lead compound to further develop the potential therapeutic anticancer agents.

Novel Podophyllotoxin Derivatives as Potential Tubulin Inhibitors: Design, Synthesis, and Antiproliferative Activity Evaluation.

A number of podophyllotoxin derivatives (3A-3J) had been designed and synthesized, and their biological activities were evaluated in this study. Moreover, the antiproliferation activities of these compounds against four human cancer cell lines (HepG2, HeLa, A549, and MCF-7) were also tested. The results indicated that the most promising compound 3D displayed potent inhibitory activity over the four human cancer cell lines and was further demonstrated to have potent tubulin polymerization inhibitory effects without damaging the non-cancer cells. Additionally, 3D was verified to effectively interfere with tubulin and could prevent the mitosis of cancer cells, leading to cell cycle arrest and eventually inducing apoptosis in a dose- and time-dependent manner. Moreover, the Western blotting and siRNA results showed that Bcl-2 was downregulated in HepG2 cells treated with 3D. Finally, the molecular docking simulation results revealed that 3D could fit well in the colchicine-binding pocket. Taken together, this study has provided certain novel antitubulin agents for possible cancer chemotherapy.

Design and characterization of α-lipoic acyl shikonin ester twin drugs as tubulin and PDK1 dual inhibitors.

Shikonin exhibits powerful anticancer activities for various cancer cells, but its poor solubility and strong toxicity hinder its development as clinical anticancer agent. We previously confirmed that shikonin and its derivatives can disturb mitosis through targeting tubulin. In this study, α-lipoic acid, the naturally-occurring co-factor of pyruvate dehydrogenase (PDH), was introduced into shikonin to design the twin drugs against both mitosis (tubulin) and glycolysis (PDK). 18 kinds of α-lipoic acid shikonin ester derivatives were achieved through three rounds of screening process performed by computer assistant drug design method, being designated as the outstanding compounds. Among them, 1c displayed the most potent cytotoxicity towards cervical cancer cells (HeLa) with an IC50 value of 3.14 ± 0.58 µM and inhibited xenotransplanted tumor growth in a dose-dependent manner. Further pharmacologic study demonstrated that 1c can cause cell cycle arrest in G2/M phase as tubulin polymerization inhibitor. Moreover, it also showed good PDK1 inhibitory activity, promoting PDH activity and forced HeLa cells to process more aerobic metabolism to undergo cell apoptosis. We reported here the first dual inhibitors of tubulin and PDK1 based on shikonin. It may form a basis for shikonin optimization through twin drug design framework for the discovery of new and potent shikonin derivatives in the study of targeted cancer therapy.

The evaluation of potent antitumor activities of shikonin coumarin-carboxylic acid, PMMB232 through HIF-1α-mediated apoptosis.

In current study, a series of shikonin derivatives were synthesized and its anticancer activity was evaluated. As a result, PMMB232 showed the best antiproliferation activity with an IC50 value of 3.25±0.35µM. Further, treatment of HeLa cells with a variety of concentrations of target drug resulted in dose-dependent event marked by apoptosis. What's more, the mitochondrial potential (Δym) analysis was consistent with the apoptosis result. In addition, PARP was involved in the progress of apoptosis revealed by western blotting. To identify the detailed role and mechanism of PMMB232 in the progression of human cervical cancer, we detected the expression of HIF-1α and E-cadherin in HeLa cells. Results showed that expression of HIF-1α was downregulated, while E-cadherin protein was upregulated. Meanwhile, glycolysis related protein PDK1 was decreased in HeLa cells. Conversely, the expression of PDH-E1α was upregulated. Docking simulation results further indicate that PMMB232 could be well bound to HIF-1α. Taken together, our data indicate that compound PMMB232 could be developed as a potential anticancer agent.

Identification of new shikonin derivatives as STAT3 inhibitors.

The signal transducer and activator of transcription 3 is a constitutively activated oncogenic protein in various human tumors and represents a valid target for anticancer drug design. In this study, we have achieved a new type of STAT3 inhibitors based on structural modifications on shikonin scaffold, guided by computational modelling. By tests, PMMB-187 exhibited a more outstanding profile than shikonin on a small panel of human breast cancer cells, especially for the MDA-MB-231 cells. For the cellular mechanisms research, PMMB-187 was found to induce cell apoptosis in MDA-MB-231 cells, associated with the reduction of mitochondrial membrane potential, production of ROS and alteration of the levels of apoptosis-related proteins. Furthermore, PMMB-187 inhibited constitutive/inducible STAT3 activation, transcriptional activity, nuclear translocation and downstream target genes expression in STAT3-dependent breast cancer cells MDA-MB-231. Besides, no obvious inhibitory effect on activation of STAT1 and STAT5 was observed with PMMB-187 treatment. Most notably, the in vivo studies further revealed that PMMB-187 could dramatically suppress the MDA-MB-231 cells xenografted tumor growth. The in vitro and in vivo results collectively suggest that PMMB-187 may serve as a promising lead compound for the further development of potential therapeutic anti-neoplastic agents.

Design and synthesis of piperazine acetate podophyllotoxin ester derivatives targeting tubulin depolymerization as new anticancer agents.

In this paper, a series of podophyllotoxin piperazine acetate ester derivatives were synthesized and investigated due to their antiproliferation activity on different human cancer cell lines. Among the congeners, C5 manifested prominent cytotoxicity towards the cancer cells, without causing damage on the non-cancer cells through inhibiting tubulin assembly and having high selectively causing damage on the human breast (MCF-7) cell line (IC50=2.78±0.15µM). Treatments of MCF-7 cells with C5 resulted in cell cycle arrest in G2/M phase and microtubule network disruption. Moreover, regarding the expression of cell cycle relative proteins CDK1, a protein required for mitotic initiation was up-regulated. Besides, Cyclin A, Cyclin B1 and Cyclin D1 proteins were down-regulated. Meanwhile, it seems that the effect of C5 on MCF-7 cells apoptosis inducing was observed to be not obvious enough. In addition, docking analysis demonstrated that the congeners occupy the colchicine binding pocket of tubulin.

Design, synthesis and anti-cancer activity evaluation of podophyllotoxin-norcantharidin hybrid drugs.

In this study, we designed and synthesized eighteen podophyllotoxin-norcantharidin hybrid drugs which could exhibit more potent anti-cancer activity than the parent drugs. Through the anti-proliferation assay, the most potent anti-cancer agent was screened out, namely Q9 (IC50=0.88±0.18µM against MCF-7 cell line), and it showed lower cytotoxicity against non-cancer cells, human embryonic kidney cells (293T) (IC50=54.38±3.78µM). Additionally, based on the flow cytometry analysis result, it can cause a remarkable cell cycle arrest at G2/M phase and induce apoptosis in MCF-7 cells more significantly than podophyllotoxin or norcantharidin per se. Moreover, the expression of cell cycle relative protein CDK1 was up regulated while a protein required for mitotic initiation, Cyclin B1 was down regulated. Furthermore, according to the confocal microscopy observation results, it was shown that Q9 was a potent tubulin polymerization inhibitor and the effect is comparable to that of colchicine. For further investigation on the aforementioned mechanisms, we performed western blot experiments, thus finding the increase of the cleavage of PARP. Consistent with these new findings, molecular docking observations suggested that compound Q9 could be developed as a potential anticancer agent.

Discovery and synthesis of a novel series of potent, selective inhibitors of the PI3Kα: 2-alkyl-chromeno[4,3-c]pyrazol-4(2H)-one derivatives.

A series of novel 2-alkyl-chromeno[4,3-c]pyrazol-4(2H)-one derivatives were synthesized and evaluated for their biological activities as PI3K inhibitors. In vitro biological evaluation against four human tumor cell lines revealed that most target compounds showed impressively better antiproliferative activities than that of LY294002. Among these compounds, compound 4l exhibited the most potent and selective activity for PI3Kα, with the value of 0.014 µM, an approximately 30-fold increase in comparison with LY294002. Docking simulation was performed to position compound 4l into the PI3Kα active site and the result showed that compound 4l could bind well at the PI3Kα active site and it indicated that compound 4l could be a potential inhibitor of PI3Kα.

Novel shikonin derivatives targeting tubulin as anticancer agents.

In this study, we report the identification of a new shikonin-phenoxyacetic acid derivative, as an inhibitor of tubulin. A series of compounds were prepared; among them, compound 16 [(R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-enyl 2-(4- phenoxyphenyl) acetate] potently inhibited the function of microtubules, inducing cell growth inhibition, apoptosis of cancer cell lines in a concentration and time-dependent manner. Molecular docking involving 16 at the vinblastine binding site of tubulin indicated that a phenoxy moiety interacted with tubulin via hydrogen bonding with asparaginate (Asn) and tyrosine (Tyr). Analysis of microtubules with confocal microscopy demonstrated that 16 altered the microtubule architecture and exhibited a significant reduction in microtubule density. Cell cycle assay further proved that HepG2 cells were blocked in G2/M phase. Our study provides a new, promising compound for the development of tubulin inhibitors by proposing a new target for the anticancer activity of shikonin.