Rujifang inhibits triple-negative breast cancer growth via the PI3K/AKT pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Rujifang (RJF) constitutes a traditional Chinese medicinal compound extensively employed in the management of triple-negative breast cancer (TNBC). However, information regarding its potential active ingredients, antitumor effects, safety, and mechanism of action remains unreported. AIM OF THE STUDY: To investigate the efficacy and safety of RJF in the context of TNBC. MATERIALS AND METHODS: We employed the ultra high-performance liquid chromatography-electrospray four-pole time-of-flight mass spectrometry technique (UPLC/Q-TOF-MS/MS) to scrutinize the chemical constituents of RJF. Subcutaneously transplanted tumor models were utilized to assess the impact of RJF on TNBC in vivo. Thirty female BLAB/c mice were randomly divided into five groups: the model group, cyclophosphamide group, and RJF high-dose, medium-dose, and low-dose groups. A total of 1 × 106 4T1 cells were subcutaneously injected into the right shoulder of mice, and they were administered treatments for a span of 28 days. We conducted evaluations on blood parameters, encompassing white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), platelet count (PLT), neutrophils, lymphocytes, and monocytes, as well as hepatorenal indicators including alkaline phosphatase (ALP), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), albumin, and creatinine (CRE) to gauge the safety of RJF. Ki67 and TUNEL were detected via immunohistochemistry and immunofluorescence, respectively. We prepared RJF drug-containing serum for TNBC cell lines and assessed the in vitro inhibitory effect of RJF on tumor cell growth through the CCK8 assay and cell cycle analysis. RT-PCR was employed to detect the mRNA expression of cyclin-dependent kinase and cyclin-dependent kinase inhibitors in tumor tissues, and Western blot was carried out to ascertain the expression of cyclin and pathway-related proteins. RESULTS: 100 compounds were identified in RJF, which consisted of 3 flavonoids, 24 glycosides, 18 alkaloids, 3 amino acids, 8 phenylpropanoids, 6 terpenes, 20 organic acids, and 18 other compounds. In animal experiments, both CTX and RJF exhibited substantial antitumor effects. RJF led to an increase in the number of neutrophils in peripheral blood, with no significant impact on other hematological indices. In contrast, CTX reduced red blood cell count, hemoglobin levels, and white blood cell count, while increasing platelet count. RJF exhibited no discernible influence on hepatorenal function, whereas Cyclophosphamide (CTX) decreased ALP, GOT, and GPT levels. Both CTX and RJF reduced the expression of Ki67 and heightened the occurrence of apoptosis in tumor tissue. RJF drug-containing serum hindered the viability of 4T1 and MD-MBA-231 cells in a time and concentration-dependent manner. In cell cycle experiments, RJF diminished the proportion of G2 phase cells and arrested the cell cycle at the S phase. RT-PCR analysis indicated that RJF down-regulated the mRNA expression of CDK2 and CDK4, while up-regulating that of P21 and P27 in tumor tissue. The trends in CDKs and CDKIs protein expression mirrored those of mRNA expression. Moreover, the PI3K/AKT pathway displayed downregulation in the tumor tissue of mice treated with RJF. CONCLUSION: RJF demonstrates effectiveness and safety in the context of TNBC. It exerts anti-tumor effects by arresting the cell cycle at the S phase through the PI3K-AKT pathway.

GPC3 and PEG10 peptides associated with placental gp96 elicit specific T cell immunity against hepatocellular carcinoma.

The placenta and tumors can exhibit a shared expression profile of proto-oncogenes. The basis of placenta-derived heat shock protein gp96, which induces prophylactic and therapeutic T cell responses against cancer including hepatocellular carcinoma (HCC), remains unknown. Here, we identified the associated long peptides from human placental gp96 using matrix-assisted laser desorption/ionization-time-of-flight and mass spectrometry and analyzed the achieved proteins through disease enrichment analysis. We found that placental gp96 binds to numerous peptides derived from 73 proteins that could be enriched in multiple cancer types. Epitope-harboring peptides from glypican 3 (GPC3) and paternally expressed gene 10 (PEG10) were the major antigens mediating anti-HCC T cell immunity. Molecular docking analysis showed that the GPC3- and PEG10-derived peptides, mainly obtained from the cytotrophoblast layer of the mature placenta, bind to the lumenal channel and client-bound domain of the gp96 dimer. Immunization with bone marrow-derived dendritic cells pulsed with recombinant gp96-GPC3 or recombinant gp96-PEG10 peptide complex induced specific T cell responses, and T cell transfusion led to pronounced growth inhibition of HCC tumors in nude mice. We demonstrated that the chaperone gp96 can capture antigenic peptides as an efficient approach for defining tumor rejection oncoantigens in the placenta and provide a basis for developing GPC3 and PEG10 peptide-based vaccines against HCC. This study provides insight into the underlying mechanism of the antitumor response mediated by embryonic antigens from fetal tissues, and this will incite more studies to identify potential tumor rejection antigens from placenta.

Strong tissue adhesive polyelectrolyte complex powders based on low molecular weight chitosan for acute hemorrhage control.

Self-gelling and bioadhesive powders offered promising effective hemostats to suit irregularly shaped, complex and non-compressible wounds for clinical applications. In the current study, chitosan based polyelectrolyte complex coacervate were simply prepared by mixing high concentrations (10 %) of low molecular weight chitosan (CS) and polyacrylic acid (PAA) solutions. Obtained by lyophilization, the physical cross-linked polyelectrolyte complex powders would form a gel within 5 s upon hydration, which demonstrated excellent mechanical properties, significant antibacterial activities, strong and lasting adhesion on wet tissues in physiological environment. In vitro blood clotting assays showed that the CS/PAA powders could remarkably aggregate blood cells and accelerate blood clotting process. As studied by diverse hemorrhage models, including rat tail, liver and heart injuries and dog incision, CS/PAA powders significantly facilitated the decrease of blood loss as well as hemostatic time by creating robust physical barriers and promoting blood clot formation on the bleeding sites. These outstanding properties in terms of easy preparation, rapid self-gelling, strong wet adhesion, effective hemostasis and shape-adaptability endowed CS/PAA polyelectrolyte complex powders with great potential in managing acute hemorrhage of non-compressible trauma.

In vivo flow cytometry reveals an anti-metastatic effect of Rujifang in triple-negative breast cancer.

Breast cancer is the most common cancer, and triple-negative breast cancer (TNBC) has the highest metastasis and mortality rate among all breast cancer subtypes. Rujifang is a traditional Chinese medicine formula with many years of clinical application in breast cancer treatment. Here, we aim to investigate the effects of Rujifang on circulating tumor cell (CTC) dynamics and the tumor microenvironment in a ZsGreen/luciferase double-labeled TNBC orthotopic model. We report that the number of CTCs monitored by in vivo flow cytometry (IVFC) strongly correlates with disease progression. Rujifang treatment decreased the number of CTCs and suppressed the distant metastasis of TNBC. Moreover, immunofluorescence analysis revealed that Rujifang treatment could affect the tumor microenvironment by downregulating Kindlin-1, which has been reported to promote metastasis of TNBC. Our study provides evidence of the anti-metastatic effect of Rujifang against TNBC in an animal model using fluorescent cell lines. The results suggest the potential therapeutic value of Rujifang as an anti-metastatic drug, however, further clinical trials are needed to validate these findings in humans.

Associations between serum soluble transferrin receptor and the prevalence of cancers.

Background: As increasing experimental evidence suggests that iron metabolism play crucial roles in cancer and non-cancer conditions, there is a lack of data on serum soluble transferrin receptor (sTfR), a promising marker representing unmet cellular iron demands, between cancer risk from epidemiological studies. Here, we aimed to evaluate the predictive value of sTfR and cancer prevalence. Materials and methods: We analyzed on 5,480 adult participants from 2015 to 2018 National Health and Nutrition Examination Survey (NHANES). Spearman correlation analysis was performed to investigate the correlations between sTfR and other characteristics. To identify the associations between sTfR and the prevalence of cancers, stratified multivariable logistic regression models, subgroup and sensitivity analyses were also performed. Results: In tertile analyses, participants in the highest level of sTfR were significantly associated with increased prevalence of total cancers [odds ratio (OR) = 1.53, 95% confidence interval (CI): 1.15-2.02] as compared with those at the lowest tertile. Each unit increment in ln-transformed sTfR concentration was shown to be associated with 39% increased risks of total cancers. Similar associations were found in males rather than females. Further subgroup and sensitivity analyses indicated that, in continuous and tertile analyses, sTfR was more closely associated with male- and female-specific cancers of prostate and testis (2.35: 1.03-5.40; 2.03: 1.00-4.09; respectively), and breast, cervix, ovary and uterus (1.92: 1.11-3.35; 1.66: 1.02-2.69; respectively). Conclusions: Our findings suggested that elevated level of sTfR was associated with the prevalence of cancers, especially in sex-specific cancers. In order to better determine them, further research in humans will be required.

A mechanistic review of chinese medicine polyphenols on bone formation and resorption.

Bone reconstruction includes a steady state system of bone formation and bone absorption. This tight coupling requires subtle coordination between osteoblasts and osteoclasts. If this balance is broken, it will lead to bone mass loss, bone density reduction, and bone metabolic diseases, such as osteoporosis. Polyphenols in Chinese herbal medicines are active ingredients in plant extracts with high safety and few side effects, and they can play a role in affecting bone formation and bone resorption. Some of these have estrogen-like effects and can better target bone health in postmenopausal women. The purpose of this review is to provide comprehensive information on the mechanisms underlying the relationship between traditional Chinese medicine polyphenols and bone formation or bone resorption.

Activating caspase-8/Bid/ROS signaling to promote apoptosis of breast cancer cells by folate-modified albumin baicalin-loaded nanoparticles.

Abnormal apoptosis can lead to uncontrolled cell growth, aberrant homeostasis or the accumulation of mutations. Therapeutic agents that re-establish the normal functions of apoptotic signaling pathways offer an attractive strategy for the treatment of breast cancer. Baicalin (BA) is one of the natural compounds with anti-proliferation and pro-apoptosis activities against numerous tumor cells. However, low bioavailability restricts the clinical application of BA. In order to improve its therapeutic efficacy and study the mechanism of actions, active targeting delivery systems were developed for targeting tumor environment and selective cell killing effects. It emphasized on the construction of folate-conjugated albumin nanoparticles loaded with baicalin (FA-BSANPs/BA) and mechanisms of which on the promotion of breast cancer apoptosis. The physicochemical properties and structural characteristics of FA-BSANPs/BA were investigated. Cell experiments were carried out to study the targeted anti-breast cancer effects of FA-BSANPs/BA and its mechanism. The results showed that FA-BSANPs/BA was successfully constructed with stable structural characteristics and sustained release effects. Cellular uptake and MTT showed that it increased targeted uptake efficiency and cytotoxicity. Flow cytometry and western blot confirmed that it promoted apoptosis by increasing the expression of caspase-8 and ROS, and decreasing the level of Bid. It is suggested that the pro-apoptotic mechanism of FA-BSANPs/BA is related to regulation of key proteins in extrinsic apoptotic pathway. In conclusion, FA-BSANPs/BA is a good delivery carrier and significantly inhibits the breast cancer growth compared with free BA. The mechanism of FA-BSANPs/BA promoting apoptosis of breast cancer may be due to its action on the caspase-8/Bid/ROS pathway.

Ginsenoside (20S)-protopanaxatriol induces non-protective autophagy and apoptosis by inhibiting Akt/mTOR signaling pathway in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) lacks a recognized therapeutic molecular target and has an unfavorable prognosis. (20S)-Protopanaxatriol (g-PPT, PPT) is an active metabolite extracted from ginseng. Accumulating evidence suggests that it has good anti-cancer activity in vivo and in vitro. In this study, we aimed to elucidate the anti-tumor effects of PPT in TNBC cells and tumor-bearing mice, as well as the relevant molecular mechanisms of autophagy and apoptosis. In vitro, we have found that PPT is capable of inducing non-protective autophagy and apoptosis, thus exerting some anti-proliferative and anti-migration activity in TNBC cells. And in vivo, the therapeutic effects of PPT were evaluated by xenograft mouse models. The potential binding mode of PPT and Akt was predicted by molecular docking. Our findings indicated that PPT treatment induced non-protective autophagy in TNBC cells by inhibiting the Akt/mTOR signaling pathway. Therefore, PPT may be a potential treatment for TNBC in the future.

Correction: Dendritic cells pulsed with placental gp96 promote tumor-reactive immune responses.

[This corrects the article DOI: 10.1371/journal.pone.0211490.].

Dendritic cells pulsed with placental gp96 promote tumor-reactive immune responses.

Defining and loading of immunogenic and safe cancer antigens remain a major challenge for designing dendritic cell (DC)-based cancer vaccines. In this study, we defined a prototype strategy of using DC-based vaccines pulsed with placenta-derived heat shock protein gp96 to induces anti-tumor T cell responses. Placental gp96 was efficiently taken up by CD11c+ bone marrow-derived DCs (BMDCs) and resulted in moderate BMDC maturation. Splenocytes and cytotoxic T cells (CTLs) generated with mouse BMDCs pulsed with placental gp96 specifically lysed B16 melanoma and LLC lung carcinoma cells. In both transplantable melanoma and lung carcinoma mice models, immunization with placental gp96-stimulated BMDCs led to a significant decrease in tumor growth and mouse mortality with respect to mice treated with liver gp96-pulsed BMDCs or placental gp96 alone. This vaccine induced strong cross-reactive tumor-specific T cell responses. Our results revealed that DCs pulsed with placenta-derived gp96 represent an effective immunotherapy to induce tumor-reactive immune responses, possibly via loading DCs with its associated carcinoembryonic antigens.

Supplementation with dietary linseed oil during peri-puberty stimulates steroidogenesis and testis development in rams.

Omega-3 polyunsaturated fatty acids (ω-3 PUFAs), such as α-linolenic acid (ALA), eicosapentaenoic acid, and docosahexaenoic acid, are involved in male reproductive function. In this study, we investigated the effects of linseed oil (LO) as a source of ALA on the steroidogenesis and changes of testicular histology in rams. Sixteen 3-month old rams during peri-puberty were randomly assigned into two groups. Eight rams were assigned as the control group, and the other received LO (4% dry matter of total diet) as the LO treatment group. After an 81-day feeding trial, the rams were slaughtered and investigated. Results revealed that compared with control group, diet containing LO did not affect body weight (36.87 ± 0.53 kg vs. 37.65 ± 0.64 kg, respectively; P = 0.361), average daily gain (227.47 ± 5.82 g vs. 237.95 ± 9.22 g, respectively; P = 0.353) and epididymis weight (40.77 ± 4.41 g vs. 45.53 ± 4.01 g, respectively; P = 0.398), however, it up-regulated PUFAs metabolism and steroidogenesis-related genes mRNA expression (P < 0.05), and increased plasma estradiol concentration (14.88 ± 0.67 pg/mL vs. 19.50 ± 1.27 pg/mL, respectively; P < 0.05). Therefore, LO stimulated seminiferous tubule development and increased the number of Sertoli cells (19.17 ± 2.14 vs. 27.2 ± 2.39, respectively; P < 0.01), germ-cell layers, as well as testis weight (148.65 ± 22.66 g vs. 249.96 ± 30.63 g, respectively; P < 0.05). All these results suggested that LO can improve testis development during peri-puberty by regulating steroidogenesis in rams' testes.

[Effects of Modified Ruji Recipe in Preventing Relapse and Metastasis of Breast Cancer Patients with Negative Hormone Receptor].

Objective To observe the effect of Ruji Recipe (RR) (treated by syndrome typing) in preventing the relapse and metastasis of invasive ductal breast cancer patients with negative hormone receptor (HR) after surgery and chemotherapy. Methods Using a prospective, cohort method, 136 pa- tients with stage I - III C HR negative invasive ductal breast cancer were equally assigned to the treat- ment group (treated by RR in syndrome typing way) and the control group (routine follow-ups). Disease free survival (DFS) , overall survival (OS) , relapse and metastasis were observed in the two groups. Re- sults All patients were followed-up for 15 to 57 months, with the median follow-up of 44 months. The median DFS and OS had not reached. The 1. 0, 1. 5, 2. -0, and 3. 0 years DFS were 94.1 % (64/68) , 86. 4 % (51/59), 81. 8% (45/55), and 72. 0% (36/50) in the treatment group. They were 77. 9% (53/68), 67.2% (45)67), 60. 6% (40)66), and 54. 5% (36/66) in the control group. Significant difference existed in 1. 0, 1. 5, and 2. 0 years DFS between the two groups (X² = 7.403, 6.426, 6.459; P =0. 012, 0.013, 0. 016). No statistical difference existed in 1. 0, 1. 5, 2. 0, and 3. 0 years OS between the two groups (P >0. 05). Among triple negative breast cancer (TNBC) patients, 1. 0, 1. 5, 2. 0, and 3. 0 years DFS were 97. 0% (32/33), 92. 9% (26/28), 92.6% (2527), and 84. 6% (22/26) in the treatment group, 81. 5% (2227), 66. 7% (1827), 61. 5% (16/26), and 57. 7% (15/26) in the control group. Of them, significant difference existed in 1. 5, 2. 0, and 3. 0 years DFS between the two groups (X² =5. 893, 7. 293, 4. 591 ; P = 0. 015, 0. 007, 0. 032). At the end of follow-ups, relapse and metastasis occurred in 15 patients, local recur- rence in 2 patients, single organ metastasis in 6 patients, and multiple organs metastasis in 7 patients of the treatment group. The relapse and metastasis occurred in 30 patients , local recurrence in 2 patients , single organ metastasis in 12 patients, and multiple organs metastasis in 16 patients of the treatment group. Conclusions RR ( by syndrome typing) could improve DFS and delay progression of invasive ductal breast cancer patients with negative HR in the first 2 years after surgery. It also had certain value for relapse and metastasis of TNBC patients within 2 years.

[Effect of ruji recipe on the post-surgical survival of female breast cancer patients].

OBJECTIVE: To observe the effect of Ruji Recipe (RR) in preventing disease recurrence/metastasis and improving quality of life (QOL) for female breast cancer patients after operation. METHODS: Totally 102 female patients with stage I - III breast cancer were retrospectively analyzed. They were assigned to the treatment group (54 cases) and the control group (48 cases) according to whether they would rather accept RR therapy. Estrogen receptor/progesterone receptor (ER/PR) positive patients also accepted endocrine therapy. The overall survival (OS), disease-free survival (DFS), recurrence and metastasis, and QOL were compared between the two groups. RESULTS: Totally 100 patients completed the study. The median follow-up was 59 months. The median OS was 60 months in the treatment group and 52.5 months in the control group (chi2 = 3.274, P > 0.05). The median DFS was 55.0 months in the treatment group and 47.5 months in the control group (chi2 = 10.145, P < 0.01). The DFS rate was 75.9% (41/54) in the treatment group and 54.3% (25/46) in the control group (chi2 = -2.259, P < 0.05). There was statistical difference in the 2-, 3-, and 5-year DFS between the two groups (P < 0.01). There was statistical difference in the 2-year DFS 3-year DFS between stage II and III and stage III (P < 0.05, P < 0.01). There was statistical difference in the ER positive patients between 2-year DFS and 3-year DFS (P < 0.01, P < 0.05). There was statistical difference in the 3-and 5-year distant metastasis rate (DMR) in the treatment group, lower than that of the control group (3.7% vs 31.0%, 20.7% vs 60.7%; P < 0.01). By the end of follow-up, disease progression occurred in 13 cases of the treatment group, local recurrence in 3 cases, single organ metastasis in 7 cases, multi-metastasis in 3 cases, while the corresponding numbers were 21, 1, 11, and 9 in the control group (P < 0.05). As for 1 week before study and at 2-year follow-up using Functional Assessment of Cancer Therapy for Breast Cancer (FACT-B) system, there was statistical difference in the QOL between the two groups (P < 0.05), and better effect was obtained in the treatment group. CONCLUSION: RR, as an assistant therapy, could improve the OS rate, the DFS rate, and the QOL for post-surgical female breast cancer patients in 2 -3 years.

Treatment of middle/late stage primary hepatic carcinoma by Chinese medicine comprehensive therapy: A prospective randomized controlled study.

OBJECTIVE: To observe the efficacy of Chinese medicine comprehensive therapeutic project in treating the middle/late stage primary hepatic carcinoma (PHC). METHODS: With prospective randomized controlled design, 97 patients with PHC were assigned to the test group (49 cases) treated with Chinese medicine comprehensive therapy using Oleum fructus bruceas intervention combining oral intake of Ganji Decoction and external application of Ailitong, and the control group (48 cases) treated with chemotherapeutic agents combining iodized oil chemo-embolization and analgesics. The immediate and long-term efficacy, adverse reaction, pain-relieving initial time (PRIT) and pain-relieving sustained time (PRST) of the treatment, as well as the change in relieving patients' quality of life (QOL) were observed. RESULTS: The difference between the two groups in illness control rate was statistically insignificant (P>0.05), but the adverse reaction occurrence rate in the test group was lesser than that in the control group (P<0.05). PRIT was insignificantly different in the two groups (P>0.05), but the PRST was significantly superior in the test group than that in the control group (10.37+/-2.18 h vs 7.78+/-1.95 h, P<0.01). After treatment, the increased Karnofsky scores in the test group indicated that the patients' somatic activity, symptoms and QOL were improved significantly, which were significantly superior to those in the control group (P<0.05). The survival rate in the two groups was similar at the 3rd month after treatment, but the test group did show superiority in terms of half- and 1-year survival rate (65.9% vs 42.5% and 38.6% vs 18.1%, respectively, P<0.05). The median survival time in the test group was 8.9 months and that in the control group was 5.3 months. CONCLUSION: Chinese medicine comprehensive therapy is an effective treatment for the middle/late stage patients of PHC, and it could extend the PRST, improve the patients' QOL and long-term survival with less adverse reaction.

[Effect of ganji recipe combined with Fructus Bruceae oil emulsion intervention on quality of life in patients with advanced primary hepatic cancer].

OBJECTIVE: To observe and compare the quality of life (QOL) and survival time in patients with advanced primary hepatic cancer (PHC) after they have been treated by the combination of ganji recipe and interventional therapy with Fructus Bruceae Oil Emulsion (FBE) or by the trans-hepatic arterial chemical embolization (TACE) adopting Seldinger's technique. METHODS: Seventy-seven patients with advanced PHC were randomly assigned to two groups, 37 patients in the control group treated with TACE alone, and 40 in the treatment group with the combined therapy. One therapeutic course was 4 weeks, and the intervention therapy was repeated generally after an interval of 4-6 weeks according to patients'condition ,the average intervention frequency was 2.3 for one case. The QOL and the survival time in patients were evaluated after treatment. RESULTS: Significant difference was shown in the changes of the Karnofsky score between the two groups (P < 0.05). The scores suggested that the improvements in the treatment group, including patients'physical energy enhancing, symptoms alleviating and overall QOL improving, were superior to those in the control group (P < 0.05). No significant difference of the 3-month survival rate between the two groups was found (P > 0.05), but the 0.5- and 1-year survival rate were significantly superior in the treatment group to those in the control group (67.6% vs. 42.4% and 38.2% vs. 16.1%, respectively). The median survival time in the two groups was 8.9 and 5.3 months respectively. CONCLUSION: Combination of ganji recipe and interventional therapy with FBE in treating PHC could improve patients' QOL, raise the 0.5- and 1-year survival rate, and prolong the survival time, suggesting TCM treatment has an affirmative significance for enhancing the therapeutic efficacy and improving the prognosis in patients.

[Effect of Fructus Bruceae oil emulsion on cellular immune function and quality of life in patients with non-small cell lung cancer].

OBJECTIVE: To observe the effect of Fructus Bruceae oil emulsion (FBE) on cellular immune function (CIF) and quality of life (QOF) in patients with non-small cell lung cancer (NSCLC) after chemotherapy. METHODS: One hundred and fifteen patients with mid-late stage NSCLC were randomly assigned to two groups, the 57 patients in the control group were only treated with chemotherapy of GP regimen, 58 in the treatment group with the chemotherapy of the same regimen and combined with FBE. The clinical efficacy was evaluated after two cycles of chemotherapy. RESULTS: The effective rate was 51.8% and 47.4% in the treatment group and the control group respectively, the difference between them was insignificant (P > 0.05). CIF and QOF in the treatment group were better than those in the control group after chemotherapy respectively (P < 0.01), in the latter, CIF and QOF were desreased after chemotherapy (P < 0.05). CONCLUSION: FBE combined with chemotherapy can improve the cellular immune function and quality of life in patients with mid-late stage NSCLC.