Combined chemo-immuno-photothermal therapy based on ursolic acid/astragaloside IV-loaded hyaluronic acid-modified polydopamine nanomedicine inhibiting the growth and metastasis of non-small cell lung cancer.

Combining chemotherapy and immunotherapy is a promising strategy for the treatment of non-small cell lung cancer (NSCLC) metastasis. However, platinum-based chemotherapeutics and immune checkpoint blockade-based cancer immunotherapy have toxic side effects and limitations. Ursolic acid (UA) and astragaloside IV (AS-IV) are natural compounds with anticancer activity sourced from Traditional Chinese medicine (TCM). However, their poor water solubilities and targeted deletions limit their medicinal value. In this study, we fabricated hyaluronic acid (HA)-modified UA/(AS-IV)-loaded polydopamine (PDA) nanomedicine (UA/(AS-IV)@PDA-HA) with a high yield at a low cost via simple synthesis. This represents a novel multifunctional nanomedicine that combines chemotherapy, photothermal therapy (PTT), and immunotherapy with an active tumor-targeting ability. The as-prepared nanomedicine not only increased the aqueous solubilities of UA and AS-IV, but also improved their active targeting abilities. HA binds specifically to the overexpressed cluster of differentiation 44 (CD44) on the surface of most cancer cells, thereby improving drug targeting. While evaluating the anticancer effect of UA/(AS-IV)@PDA-HA in vitro and in vivo, the PDA nanodelivery system significantly improved UA-mediated cytotoxicity and anti-metastatic ability against NSCLC cells. In addition, the system also improved the AS-IV-mediated self-immune response of tumor-related antigens, which further inhibited the growth and distant metastasis of NSCLC. Further, PDA nanomaterial-mediated PTT inhibited tumor growth substantially. UA/(AS-IV)@PDA-HA not only significantly eradicated the primary tumor but also strongly inhibited the distant metastasis of NSCLC in vitro and in vivo. Thus, it has immense potential for development as an efficient anti-metastatic agent for NSCLC.

Characteristic of immune function decline in the process of “Declined of Vital Qi” caused by aging in mice / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：基于小鼠渐进衰老模型探讨衰老所致“正虚”的免疫功能衰退表征的特点。方法：使用不同月龄（2、6、15月龄）C57BL/6小鼠，通过流式细胞术检测并比较小鼠外周血和脾组织中T细胞、髓源性抑制细胞（MDSC）及其亚群的丰度变化。结果：外周血中T细胞亚群表型为CD3+CD4+CD44-CD62L+的幼稚CD4+ T细胞（2 vs 6月龄，P=0.137；2 vs 15月龄，P=0.004；6 vs 15月龄，P=0.105）和表型为CD3+CD8+CD44-CD62L+的幼稚CD8+ T细胞（2 vs 6月龄，P=0.179；2 vs 15月龄，P=0.001；6 vs 15月龄，P=0.015）出现与衰老有关的细胞比例降低，差异具有统计学意义。表型为CD3+CD4+CD44+CD62L+的中央记忆CD8+ T细胞出现与衰老有关的比例升高，差异具有统计学意义（2 vs 6月龄，P=0.01；2 vs 15月龄，P=0.007；6 vs 15月龄，P=0.164）。对脾组织的检测结果具有与外周血相同特点。同时，CD8+ T细胞比例随衰老逐渐升高（2 vs 6月龄，P=0.027；2 vs 15月龄，P<0.001；6 vs 15月龄，P<0.001）；表型为CD8+CD28+的活化CD8+ T细胞亚群比例也出现随月龄增长的上升（2 vs 6月龄，P=0.863； 2 vs 15月龄，P=0.016；6 vs 15月龄，P=0.024），差异均具有统计学意义。结论：衰老所致“正虚”过程中，不同免疫细胞亚群变化并不都反映免疫抑制特点，虽然总体免疫功能下降，但单一表型难以反应整体免疫功能变化。

Establishment of a Nomogram-Based Prognostic Model (LASSO-COX Regression) for Predicting Progression-Free Survival of Primary Non-Small Cell Lung Cancer Patients Treated with Adjuvant Chinese Herbal Medicines Therapy: A Retrospective Study of Case Series.

Nowadays, Jin-Fu-Kang oral liquid (JFK), one of Chinese herbal medicines (CHMs) preparations, has been widely used as an adjuvant therapy for primary non-small cell lung cancer (PNSCLC) patients with the syndrome of deficiency of both Qi and Yin (Qi-Yin deficiency pattern) based on Traditional Chinese Medicine (TCM) theory. However, we found insufficient evidence of how long-term CHM treatment influence PNSCLC patients' progression-free survival (PFS). Thus, using electronic medical records, we established a nomograph-based prognostic model for predicting PNSCLC patients' PFS involved with JFK supplementary formulas (JFK-SFs) over 6 months, in order to preliminarily investigate potential predictors highly related to adjuvant CHMs therapies in theoretical epidemiology. In our retrospective study, a series of 197 PNSCLC cases from Long Hua Hospital were enrolled by non-probability sampling and divided into 2 datasets at the ratio of 5:4 by Kennard-Stone algorithm, as a result of 109 in training dataset and 88 in validation dataset. Besides, TNM stage, operation history, sIL-2R, and CA724 were considered as 4 highly correlated predictors for modeling based on LASSO-Cox regression. Additionally, we respectively used training dataset and validation dataset for establishment including internal validation and external validation, and the prediction performance of model was measured by concordance index (C-index), integrated discrimination improvement, and net reclassification indices (NRI). Moreover, we found that the model containing clinical characteristics and bio-features presented the best performance by pairwise comparison. Next, the result of sensitivity analysis proved its stability. Then, for preliminarily examination of its discriminative power, all eligible cases were divided into high-risk or low-risk progression by the cut-off value of 57, in the light of predicted nomogram scores. Ultimately, a completed TRIPOD checklist was used for self-assessment of normativity and integrity in modeling. In conclusion, our model might offer crude probability of uncertainly individualized PFS with long-term CHMs therapy in the real-world setting, which could discern the individuals implicated with worse prognosis from the better ones. Nevertheless, our findings were prone to unmeasured bias caused by confounding factors, owing to retrospective cases series.

Jinfukang inhibits lung cancer metastasis by upregulating CX3CL1 to recruit NK cells to kill CTCs.

ETHNOPHARMACOLOGICAL RELEVANCE: Circulating tumor cells (CTCs) play an important role in tumor metastasis and may be a target for metastasis prevention. The traditional Chinese medicine Jinfukang functions to improve immunity, prevent metastasis, and prolong lung cancer patient survival periods. Yet, whether Jinfukang prevents metastasis by regulating immune cells to clearance CTCs is still unknown. AIM OF THE STUDY: To explore the anti-metastasis mechanism of Jinfukang from the perspective of regulating NK cells to clear CTCs. MATERIALS AND METHODS: CTC-TJH-01 cell was treated with Jinfukang. Cytokine chip was used to detect cytokines in cell culture supernatant. Lymphocyte recruitment assay was detected by Transwell and flow cytometry. Protein expression was analysis by Western blot. LDH kit was used to detect cytotoxicity. NOD-SCID mice used for tail vein injection to study lung metastasis. RESULTS: Jinfukang could promote the expression and secretion of the chemokine CX3CL1 by CTCs. In addition, Jinfukang could promote the recruitment of natural killer (NK) cells by CTCs and significantly increase the cytotoxic effect of NK cells on CTCs. Moreover, Jinfukang could upregulate the expression of FasL and promote the secretion of TNF-α by NK cells and that NK cells could induce the apoptosis of CTCs through the Fas/FasL signaling pathway. Finally, we confirmed that Jinfukang could promote NK cells to kill CTCs and then inhibit lung cancer metastasis in vivo. The above effects of Jinfukang could be partially reversed by an anti-CX3CL1 mAb. CONCLUSIONS: These results suggest that Jinfukang may prevent lung cancer metastasis by enhancing the clearance of CTCs in the peripheral blood by NK cells, providing evidence for the anti-metastasis effect of Jinfukang.

Molecular Mechanism of Bushen Jianpi Inhibition of Postoperative Recurrence and Metastasis of Hepatocellular Carcinoma.

Compared with western medicine, traditional Chinese medicine can better regulate the internal environment and inhibit liver cancer recurrence and metastasis. Bushen Jianpi Recipe (BSJPR) is a traditional Chinese medicine for tonifying the kidney and invigorating the spleen. It has also been used to treat tumors and other related diseases. Here we explore the efficacy of BSJPR inhibition of hepatocellular carcinoma (HCC) in vivo and in vitro . We hypothesize that BSJPR reduces intrahepatic cholestasis and inflammation and increases expression of the bile acid receptor and downstream targets. This study aims to test this hypothesis and determine whether the inhibitory effect of BSJPR on liver cancer recurrence and metastasis is related to bile acid metabolism. We also observed changes in immune cell expression, suggesting that regulation of the immune microenvironment could inhibit the recurrence and metastasis of HCC. These findings provide a basis for the treatment of HCC and new ideas for follow-up studies of BSJPR.

Jinfukang regulates integrin/Src pathway and anoikis mediating circulating lung cancer cells migration.

ETHNOPHARMACOLOGICAL RELEVANCE: Metastasis is the main cause of death in lung cancer patients. Circulating tumor cells (CTCs) may be an important target of metastasis intervention. Previous studies have shown that Jinfukang could prevent the recurrence and metastasis of lung cancer, and we have established a circulating lung tumor cell line CTC-TJH-01. However, whether Jinfukang inhibition of lung cancer metastasis is related to CTCs is still unknown. AIM OF THE STUDY: To further explore the mechanism of Jinfukang in anti-metastasis of lung cancer from the perspective of intervention of CTCs. MATERIALS AND METHODS: CTC-TJH-01 and H1975 cells were treated with Jinfukang. Cell viability was detected by CCK8, and the cell apoptosis was detected by flow cytometry. Transwell was used to detected cell migration and invasion. Cell anoikis was detected by anoikis detection kit. Protein expression was analysis by Western blot. RESULTS: Jinfukang could inhibit the proliferation, migration and invasion of CTC-TJH-01 and H1975 cells. Besides, Jinfukang could also induce anoikis in CTC-TJH-01 and H1975 cells. Analysis of the mRNA expression profile showed ECM-receptor interaction and focal adhesion were regulated by Jinfukang. Moreover, it was also find that Jinfukang significantly inhibited integrin/Src pathway in CTC-TJH-01 and H1975 cells. When suppress the expression of integrin with ATN-161, it could promote Jinfukang to inhibit migration and induce anoikis in CTC-TJH-01 and H1975 cells. CONCLUSIONS: Our results indicate that the migration and invasion of CTCs are inhibited by Jinfukang, and the mechanism may involve the suppression of integrin/Src axis to induce anoikis. These data suggest that Jinfukang exerts anti-metastatic effects in lung cancer may through anoikis.

α­Solanine inhibits growth and metastatic potential of human colorectal cancer cells.

Solanum nigrum L. (Longkui) is one the most widely used anticancer herbs in traditional Chinese medicine. α­Solanine is an important ingredient of S. nigrum L. and has demonstrated anticancer properties in various types of cancer. However, the effects of α­solanine on colorectal cancer remain elusive. The aim of the present study was to assess the effects of α­solanine on human colorectal cancer cells. The results demonstrated that α­solanine inhibited the proliferation of RKO cells in a dose­ and time­dependent manner. In addition, α­solanine arrested the cell cycle at the G0/G1 phase and suppressed the expression levels of cyclin D1 and cyclin­dependent kinase 2 in RKO cells. α­Solanine induced apoptosis of RKO cells, as indicated by morphological changes and positive Annexin­FITC/propidium iodide staining. Additionally, α­solanine activated caspase­3, ­8 and ­9 in RKO cells, which contributed to α­solanine­induced apoptosis. α­Solanine also increased the generation of reactive oxygen species, which contributed to caspase activation and induction of apoptosis. α­Solanine inhibited the migration, invasion and adhesion of RKO cells, as well as the expression levels and activity of matrix metalloproteinase (MMP)­2 and MMP­9. In addition, α­solanine inhibited cell proliferation, activated caspase­3, ­8 and ­9, induced apoptosis, and inhibited the migration and invasion of HCT­116 cells. Furthermore, α­solanine inhibited tumor growth and induced apoptosis in vivo. These findings demonstrated that α­solanine effectively suppressed the growth and metastatic potential of human colorectal cancer.

Proteomics analysis of tumor exosomes reveals vital pathways of Jinfukang inhibiting circulating tumor cells metastasis in lung cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: Jinfukang has long been used for the clinical treatment of lung cancer. Previous studies have shown that Jinfukang can induce the apoptosis of circulating tumor cells by intervening ROS-mediated DNA damage pathway. However, whether Jinfukang can inhibit the metastasis of circulating tumor cells and its mechanism are still unclear. AIM OF THE STUDY: To further investigate the mechanism of Jinfukang in anti-metastasis of lung cancer from the perspective of intervention of tumor exosomes. MATERIALS AND METHODS: The invadopodia formation was determined with immunofluorescence. Invasion and migration were detected using the Transwell assay. Ultracentrifugation was used to isolate exosomes. Exosomes were characterized by electron microscopy, nanoparticle tracking analysis and immunoblotting, and the protein profile was evaluated by proteomic analysis. The molecular functions, biological processes and signaling pathway enrichment analysis were performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Key differentially expressed proteins were verified by Western blot. RESULTS: Jinfukang can inhibit the expression of MMP14, cortactin, Tks5 and the formation of invadopodia of CTC-TJH-01 cells. Furthermore, Jinfukang can significantly inhibit the invasion and migration of CTC-TJH-01 cells. The diameter of exosomes extracted from the CTC-TJH-01 cells treated by Jinfukang was 30-100 nm, and the exosomal markers CD63, CD81 and TSG101 were expressed. We identified 680 deferentially expressed proteins. Gene oncology analysis indicated that exosomes were mostly derived from plasma membrane and mainly involved in protein localization and intracellular signaling. The ingenuity pathway analysis showed that the EGF pathway was significantly inhibited, whereas the GP6 signaling pathway was significantly activated. We also confirmed that Jinfukang inhibited the expression of EGF pathway-related proteins in CTC-TJH-01 cells. Besides, when EGF was used to activate EGF signaling pathway, the inhibition of Jinfukang on CTC cell metastasis was reversed. CONCLUSION: Jinfukang inhibits the metastasis of CTC-TJH-01 cells through the EGF pathway.

Jingfukang induces anti-cancer activity through oxidative stress-mediated DNA damage in circulating human lung cancer cells.

BACKGROUND: Metastasis is the main cause of lung cancer death. As a seed of metastasis, circulating tumor cells are an important target for metastasis intervention. The traditional Chinese medicine, Jinfukang, has been clinically available for the treatment of non-small cell lung cancer (NSCLC). In this study, we investigated the action and underlying mechanisms of Jinfukang against circulating lung tumor cells. METHODS: The cell counting kit-8 (CCK-8), colony formation and cell cycle assays were used to study the cell proliferation ability. Flow cytometry was used to detect the apoptosis and the expression level of ROS and Caspase-3. Comet and TUNEL assays were used to detect DNA damage. DNA damage related pathway protein was detected by western blot. RESULTS: Jinfukang significantly inhibits the proliferation of CTC-TJH-01 cells by inducing G1 phase arrest and inhibits their colony formation in a dose-dependent manner. Moreover, Jinfukang induces apoptosis in CTC-TJH-01 cells through the ROS-mediated ATM/ATR-p53 pathway and DNA damage. CONCLUSIONS: Our findings suggest that Jinfukang may be a potential drug for lung cancer metastasis.

Feiji Recipe inhibits the growth of lung cancer by modulating T-cell immunity through indoleamine-2,3-dioxygenase pathway in an orthotopic implantation model.

OBJECTIVE: Escape from the body's immune response is a basic characteristic of lung cancer, and indoleamine-2,3-dioxygenase (IDO) plays a key role in mediating immune escape of non-small-cell lung cancer, which leads to recurrence and metastasis. Feiji Recipe, a compound Chinese herbal medicine, has the effect of stabilizing lesions and prolonging survival in patients with lung cancer. The purpose of this study was to investigate the mechanisms underlying the anticancer properties of Feiji Recipe. METHODS: An orthotopic transplant model of mouse Lewis lung cancer, with stable expression of IDO gene, was established in C57BL/6 mice. Optical imaging was used to observe the effects of Feiji Recipe in the treatment of lung cancer in vivo. The effects of Feiji Recipe on the proliferation of mouse Lewis lung cancer cell line 2LL, 2LL-enhanced green fluorescent protein (2LL-EGFP) and 2LL-EGFP-IDO were investigated, and the apoptosis of T-cells was examined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide using flow cytometry. Chemical composition of Feiji Recipe was validated by high-performance liquid chromatography. RESULTS: Compared to the control group, the survival of animals treated with Feiji Recipe was significantly prolonged (P = 0.0074), and the IDO protein level decreased (P = 0.0072); moreover, the percentages of CD4+CD25+ T-cells and Foxp3+ T-cells were significantly decreased (P < 0.05). The molecular mechanism of Feiji Recipe against lung cancer may relate to the regulation of immune cells, such as T-cells and regulatory T-cells. CONCLUSION: The molecular mechanism of Feiji Recipe in treatment of lung cancer is to restore the function of T-cells in the cancer microenvironment through interfering with the IDO pathway.

Herbal formula YYJD inhibits tumor growth by inducing cell cycle arrest and senescence in lung cancer.

Lung cancer represents a major cause of cancer-related death worldwide. Although various tactics and anti-tumor drugs have been used to improve curative effects, five-year survival rate of lung cancer patients remains poor. In this study, we investigated the action and underlying mechanisms of our recently optimized Chinese herbal formula Yangyinjiedu (YYJD) against lung cancer. YYJD significantly inhibits the proliferation of lung cancer cell lines (95-D, A549, H460 and H1975) by inducing cell cycle arrest and senescence in a dose-dependent manner. In particular, YYJD induces significant G2/M phase arrest and inhibits the colony formation of lung cancer cells. Moreover, we found that administration of YYJD could inhibit the growth of xenografted lung cancer cells in nude mice without loss in body weight. Our findings suggest that the herbal formula YYJD is a potential anti-tumor agent against lung cancer.

Ganoderic acid Me induces the apoptosis of competent T cells and increases the proportion of Treg cells through enhancing the expression and activation of indoleamine 2,3-dioxygenase in mouse lewis lung cancer cells.

The indoleamine 2,3-dioxygenase-(IDO-) mediated microenvironment plays an important role in tumor immune escape. It is known that ganoderic acid Me can enhance IFN-γ expression and IDO is preferentially induced by IFN-γ. However, whether GA-Me can induce IDO expression has not been clarified yet. We established stable clones of IDO-overexpressing 2 LL cells (2LL-EGFP-IDO). After co-culturing with IDO expressing or control vector-transfected 2LL-EGFP cells, T cell apoptosis was determined and the proportion of the regulatory T cells (Tregs) and CD8+ T cell subset was measured. The total cellular protein samples of 2 LL-EGFP-IDO cells were isolated for detecting JAK-STAT1 signalling pathway. Co-culture supernatants were used to detect amino acids and cytokines. IDO transfected 2 LL cells yielded high level of IDO enzymatic activity, resulting in complete depletion of tryptophan from the culture medium. We found that apoptosis occurred in T cells after cocultured with IDO+2LL cells and the proportion of CD4+CD25+ cells and FoxP3+ cells increased while CD8+ cells decreased. The specific inhibitor of IDO, 1-D-MT and GA-Me efficiently enhanced T cell apoptosis, increased Tregs, and reduced CD8+ T cells in vitro. Increased expression of IDO, p-JAK1 and p-STAT1 were confirmed by Western blot analysis. The levels of IFN-γ, IL-10, LDH and kynurenine in co-culture supernatant correspondingly increased, while tryptophan reduced. These results suggest that GA-Me contributing to IDO helps to create a tolerogenic milieu in lung tumors by directly inducing T cell apoptosis, restraining CD8+ T cell activation, and enhancing Treg-mediated immunosuppression.

Astragaloside IV inhibits progression of lung cancer by mediating immune function of Tregs and CTLs by interfering with IDO.

PURPOSE: Tumor cells have developed multiple mechanisms to escape immune recognition mediated by T cells. Indoleamine 2,3-dioxygenase (IDO), a tryptophan-catabolizing enzyme inducing immune tolerance, is involved in tumor escape from host immune systems in mice. Astragaloside IV (AS-IV), an extract from a commonly used Chinese medicinal plant Astragalus membranaceus, has been shown to be capable of restoring the impaired T-cell functions in cancer patients. The purpose of this study was to investigate the mechanisms underlying the anticancer properties of AS-IV. METHODS: Here, we used IDO-overexpressed murine Lewis lung carcinoma cells to establish an orthotopic lung cancer model in C57BL/6 mice. Next, tumor growth was evaluated in several different treatment groups: control (saline), AS-IV, paclitaxel, and 1-methyl tryptophan (an inhibitor of IDO). We then analyzed the percentages of various immune cell subsets among the splenic lymphocytes of lung cancer mice by flow cytometry. The level of IDO was measured by real-time PCR and Western blot. RESULTS: We showed that the growth of tumor was suppressed by AS-IV treatment in vivo. AS-IV also could down-regulate regulatory T cells (Tregs) and up-regulate cytotoxic T lymphocytes (CTLs) in vivo and in vitro. Consistent with its ability to interfere with T-cell immunity, AS-IV blocked IDO induction both in vitro and in vivo. CONCLUSIONS: The results of these studies indicate that AS-IV has in vivo anticancer activity and can enhance the immune response by inhibiting the Tregs frequency and induce the activity of CTLs, which might be related to the inhibition of IDO expression.