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Shikonin-treated 4 T1 tumor cell lysate effectively activates DCs that in turn induce T-cell proliferation in vitro. SK-TCL or Dox-TCL samples were prepared from transgenic Luc2-labelled 4 T1 cells that were treated with shikinon or doxorubicin at 5 μM for 24 h. Mouse bone marrow-derived DCs were then treated with Naive-, SK- or Dox-TCL samples and used as stimulator cells. Splenic CD4 + and CD8 + T cells were collected from syngeneic mice and employed as responder cells. Proliferation activities of a CD4 + and b CD8 + T cells were analyzed by mixed lymphocyte reaction (MLR) assay. Ratios of stimulator to responder cells were set between 1:1000 and 1:5. T-cell proliferation activity is represented as fold change over the control (T cells only). Data represent the mean ± SD of three biological replicates, and an independent experiment showed similar results
Source publication
Background:
The tumor cell lysate-pulsed, dendritic cell (DC)-based cancer vaccine approaches are being actively evaluated for application to cancer immunotherapy, hopefully at a personalized medicine base. There is apparently an emerging technical problem however, the lack of highly efficacious potency in activation of patient's DCs for T-cell pr...
Contexts in source publication
Context 1
... in culture with shikonin-treated 4 T1 tumor cell lysates (SK-TCL), and the effect of the resultant DCs on T- cell proliferation was tested by MLR assay. As compared to treatment with mDCs only, all TCL-treated mDC groups, including Naive-TCL, SK-TCL and Dox-TCL, were found to stimulate dose-dependent proliferation of both CD4 + and CD8 + cells (Fig. 1a and 1b, respectively) at a DC/T cell ratio between 1:1000 and 1:5. At the ratio of 1:5, when compared to mDC group, the SK-TCL group was found to significantly induce CD4 + and CD8 + T-cell proliferation up to 5.27-and 3.53-fold (Fig. 1a and 1b), respectively, as calculated by the formula [(SK-TCL/iDC)-(mDC/iDC)]/ [(Naive-TCL/iDC)-(mDC/iDC)]. ...
Context 2
... SK-TCL and Dox-TCL, were found to stimulate dose-dependent proliferation of both CD4 + and CD8 + cells (Fig. 1a and 1b, respectively) at a DC/T cell ratio between 1:1000 and 1:5. At the ratio of 1:5, when compared to mDC group, the SK-TCL group was found to significantly induce CD4 + and CD8 + T-cell proliferation up to 5.27-and 3.53-fold (Fig. 1a and 1b), respectively, as calculated by the formula [(SK-TCL/iDC)-(mDC/iDC)]/ [(Naive-TCL/iDC)-(mDC/iDC)]. In comparison, Dox-TCL exhibited only a 3.47-and 2.40-fold change in CD4 + and CD8 + T-cell proliferation activities, as shown in Fig. 1a and 1b, respectively. These results suggest that SK-TCL- activated mDCs can effectively induce the ...
Context 3
... group was found to significantly induce CD4 + and CD8 + T-cell proliferation up to 5.27-and 3.53-fold (Fig. 1a and 1b), respectively, as calculated by the formula [(SK-TCL/iDC)-(mDC/iDC)]/ [(Naive-TCL/iDC)-(mDC/iDC)]. In comparison, Dox-TCL exhibited only a 3.47-and 2.40-fold change in CD4 + and CD8 + T-cell proliferation activities, as shown in Fig. 1a and 1b, respectively. These results suggest that SK-TCL- activated mDCs can effectively induce the proliferation of both CD4 + and CD8 + T cells. These activities were not only significantly higher than the Naive-TCL group, they were even moderately higher than those obtained from the Dox- TCL ...
Context 4
... results outlined above and shown in Fig. 1 suggest that SK-TCL may possess potential for augmenting T cell-mediated anti-tumor activity. We therefore next evaluated the efficacy of use of Naive-TCL, SK-TCL or Dox-TCL as adjuvants for a DC vaccine formulation aiming to inhibit tumor metastasis in a mouse 4 T1 mammary tumor-resection model. A bioluminescent, transgenic ...
Context 5
... one of our previous studies, we demonstrated that shikonin can effectively induce the expression of specific DAMPs in treated tumor cells, and these DAMPs can in turn activate a cascade of caspase activities, which can, in combination, activate DCs to a full level of maturation and activation, resulting in potent Th17 and Th1 cell activities that medi- ate efficacious retardation of tumor growth and prolong the survival of test mice [8]. In this study, among various ICD-related protein components, both HSP70 and CRT were demonstrated to play an essential role in SK-TCL- induced DC immunity by stimulating both CD4 + and CD8 + T cell proliferation (Figs. 1 and 4). Our in vivo result is not in agreement, or perhaps even contradicts, reports that HMGB1 is the most vital determinant of the im- munogenicity of ICD to DCs [20,21]. ...
Citations
... Instigated autophagy promotes DAMP ecto-localization [83]. Autophagy-induced ectoDAMPs have been proposed as effective compounds for activating tumor cell lysate (TCL)-pulsed dendritic cell (DC)-based vaccines [84]. To investigate other mechanisms, Yin et al. revealed that shikonin suppresses the function of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) and controls hnRNPA1-targeted gene splicing activities. ...
... In mouse models of BRCA, SK-TCL-activated DC vaccine injections resulted in higher survival rates and a lower incidence of metastasis. More interestingly, SK-TCL was more effective than doxorubicin (Dox)-TCL in activating DC vaccines and exerting anticancer effects [84]. ...
Objectives
Breast cancer is a prevalent disease that has a substantial impact on women’s mortality rates. Shikonin, a naphthoquinone derived from Lithospermum erythrorhizon, has demonstrated substantial anticancer effects. This study aims to conduct a comprehensive review of the latest research findings regarding the therapeutic efficacy of shikonin in the context of breast cancer treatment, with a specific emphasis on elucidating the underlying molecular mechanisms.
Methods
A comprehensive literature review was conducted on shikonin and breast cancer by searching PubMed, Scopus, Web of Science, and Google Scholar databases.
Key findings
Shikonin significantly reduces tumor cell viability, proliferation, migration, invasion, and metastasis in both in vivo and in vitro across all breast cancer subtypes. Additionally, when combined with other pharmaceutical agents, it exhibits synergistic effects. Shikonin stimulates immunogenic cell death, resulting in apoptosis and necroptosis. The induction of immunogenic cell death by shikonin enhances the immunogenicity of breast cancer cells, leading to its involvement in the development of dendritic cell-based tumor vaccines against breast cancer.
Conclusion
Shikonin exhibits potent anti-breast cancer properties and shows significant potential for the advancement of immunotherapeutic approaches against breast cancer, as well as enhancing the efficacy of conventional treatment strategies.
... Shikonin (SHK), a PKM2 inhibitor with antitumor efficiency, is also known to induce ICD through binding and interfering with its heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), triggering necroptosis and ROS generation thus eliciting antitumor immunity and promoting DC maturation 54 . Therefore, SHK was encapsulated into various types of nanomedicines to enhance ICD power (Fig. 2). ...
Resistance to cancer immunotherapy is mainly attributed to poor tumor immunogenicity as well as the immunosuppressive tumor microenvironment (TME) leading to failure of immune response. Numerous therapeutic strategies including chemotherapy, radiotherapy, photodynamic photothermal, magnetic, chemodynamic, sonodynamic and oncolytic therapy, have been developed to induce immunogenic cell death (ICD) of cancer cells and thereby elicit immunogenicity and boost the antitumor immune response. However, many challenges hamper the clinical application of ICD inducers resulting in modest immunogenic response. Here, we outline the current state of using nanomedicines for boosting ICD of cancer cells. Moreover, synergistic approaches used in combination with ICD inducing nanomedicines for remodeling the TME via targeting immune checkpoints, phagocytosis, macrophage polarization, tumor hypoxia, autophagy and stromal modulation to enhance immunogenicity of dying cancer cells were analyzed. We further highlight the emerging trends of using nanomaterials for triggering amplified ICD-mediated antitumor immune responses. Endoplasmic reticulum localized ICD, focused ultrasound hyperthermia, cell membrane camouflaged nanomedicines, amplified reactive oxygen species (ROS) generation, metallo-immunotherapy, ion modulators and engineered bacteria are among the innovative approaches. Various challenges, merits and demerits of ICD inducer nanomedicines were also discussed with shedding light on the future role of this technology in improving the outcomes of cancer immunotherapy.
... Lin et al. has found that Shikonin can enhance the cellular immunogenicity of tumor vaccines through different DAMPs. Among three DAMPs tested, they found that the Hsp70 is the most important component in facilitating DC immunity on inhibiting metastasis of mouse tumors and prolonging mouse survival [18]. However, the complete understanding of the mechanism behind Hsp70 upregulation induced by Shikonin is still under investigation. ...
... They also found that CRT and Hsp70 mediated a critical role in Shikonin-treated 4T1 cell lysates-induced DC cell immunity, with significant CD4 + and CD8 + T cell proliferation. Besides, Hsp70 is regarded as the most critical component in enhancing DC immunity on suppressing tumor metastasis [18]. In our study, the upregulation of Hsp70 in tumor tissue was also observed after SK and PD-1 blockade treatments. ...
Background
PD-1 blockade has shown impressive clinical outcomes in colorectal cancers patients with high microsatellite instability (MSI-H). However, the majority of patients with colorectal cancer who present low microsatellite instability (MSI-L) or stable microsatellites (MSS) show little response to PD-1 blockade therapy. Here, we have demonstrated that Shikonin (SK) could induce cell death of CT26 cells via classically programmed and immunogenic pathways.
Methods and results
SK promoted the membrane exposure of calreticulin and upregulated the expression of heat shock protein 70 (Hsp70). The upregulation of Hsp70 was dependent on ROS induced by SK and silencing of PKM2 in CT26 cells reverts ROS upregulation. Besides, SK synergizes with PD-1 blockade in CT26 tumor mice model, with the increase of intramural DC cells and CD8+ T cells. The expression of Hsp70 in tumor tissue was also increased in combinational SK plus αPD-1 therapy group.
Conclusions
Our study elucidated the potential role of ‘Shikonin-PKM2-ROS-Hsp70’ axis in the promotion of efficacy of PD-1 blockade in CRC treatments, providing a potential strategy and targets for improving the efficacy of PD-1 blockade in colorectal cancer.
... Lin et al. provided evidence that shikonin can effectively induce ICD, and this effect may serve as an adjuvant for use in DC-based cancer vaccines. In other words, shikonin can enhance the immunogenicity of vaccines via ICD [84]. ...
A growing body of evidence indicates that the anticancer effect of the immune system can be activated by the immunogenic modulation of dying cancer cells. Cancer cell death, as a result of the activation of an immunomodulatory response, is called immunogenic cell death (ICD). This regulated cell death occurs because of increased immunogenicity of cancer cells undergoing ICD. ICD plays a crucial role in stimulating immune system activity in cancer therapy. ICD can therefore be an innovative route to improve anticancer immune responses associated with releasing damage-associated molecular patterns (DAMPs). Several conventional and chemotherapeutics, as well as preclinically investigated compounds from natural sources, possess immunostimulatory properties by ICD induction. Natural compounds have gained much interest in cancer therapy owing to their low toxicity, low cost, and inhibiting cancer cells by interfering with different mechanisms, which are critical in cancer progression. Therefore, identifying natural compounds with ICD-inducing potency presents agents with promising potential in cancer immunotherapy. Naturally derived compounds are believed to act as immunoadjuvants because they elicit cancer stress responses and DAMPs. Acute exposure to DAMP molecules can activate antigen-presenting cells (APCs), such as dendritic cells (DCs), which leads to downstream events by cytotoxic T lymphocytes (CTLs) and natural killer cells (NKs). Natural compounds as inducers of ICD may be an interesting approach to ICD induction; however, parameters that determine whether a compound can be used as an ICD inducer should be elucidated. Here, we aimed to discuss the impact of multiple ICD inducers, mainly focusing on natural agents, including plant-derived, marine molecules, and bacterial-based compounds, on the release of DAMP molecules and the activation of the corresponding signaling cascades triggering immune responses. In addition, the potential of synthetic agents for triggering ICD is also discussed.
... Shikonin was demonstrated to be an adjuvant for DC-based cancer vaccines via inducing ICD with upregulation of HSP70 and translocation of CRT. Dying tumor cells could further promote the maturation of DCs and activate Th1 cells.116,117 Oleandrin, a cardiac glycoside, triggered endoplasmic reticulum (ER) stress and inducing CRT exposure, and release of HMGB1, HSP70/90, and ATP in breast cancer cells. ...
Immunogenic cell death (ICD) is one of the 12 distinct cell death forms, which can trigger immune system to fight against cancer cells. During ICD, a number of cellular changes occur that can stimulate an immune response, including the release of molecules called damage‐associated molecular patterns (DAMPs), signaling to immune cells to recognize and attack cancer cells. By virtue of their pivotal role in immune surveillance, ICD‐based drug development has been a new approach to explore novel therapeutic combinations and personalized strategies in cancer therapy. Several small molecules and microbes can induce ICD‐relevant signals and cause cancer cell death. In this review, we highlighted the role of microbe‐mediate ICD in cancer immunotherapy and described the mechanisms through which microbes might serve as ICD inducers in cancer treatment. We also discussed current attempts to combine microbes with chemotherapy regimens or immune checkpoint inhibitors (ICIs) in the treatment of cancer patients. We surmise that manipulation of microbes may guide personalized therapeutic interventions to facilitate anticancer immune response.
... They also found that CRT and Hsp70 mediated a critical role in Shikonin-treated 4T1 cell lysates-induced DC cell immunity, with signi cant CD4 + and CD8 + T cell proliferation. Besides, Hsp70 is regarded as the most critical component in enhancing DC immunity on suppressing tumor metastasis [31]. In our study, the upregulation of Hsp70 in tumor tissue was also observed after SK and PD-1 blockade treatments. ...
Background: PD-1 blockade has shown impressive clinical outcomes in colorectal cancers patients with high microsatellite instability (MSI-H). However, the majority of patients with colorectal cancer who present low microsatellite instability (MSI-L) or stable microsatellites (MSS) show little response to PD-1 blockade therapy. Here, we have demonstrated that Shikonin (SK) could induce cell death of CT26 cells via classically programmed and immunogenic pathways.
Methods and Results: SK promoted the membrane exposure of calreticulin and upregulated the expression of heat shock protein 70 (Hsp70). The upregulation of Hsp70 was dependent on ROS induced by SK and silencing of PKM2 in CT26 cells reverts ROS upregulation. Besides, SK synergizes with PD-1 blockade in CT26 xenograft mice model, with the increase of intramural DC cells and CD8⁺T cells. The expression of Hsp70 in tumor tissue was also increased in combinational SK plus αPD-1 therapy group.
Conclusions: Our study elucidated the potential role of ‘Shikonin-PKM2-ROS-Hsp70-ICD’ axis in the promotion of efficacy of PD-1 blockade in CRC treatments, providing a potential strategy and targets for improving the efficacy of PD-1 blockade in colorectal cancer.
... SHK can induce immunogenic cell death (ICD) which triggers damage-associated molecular patterns (DAMPs) that are characterized by calreticulin (CRT) eversion and the release of high mobility group box 1 (HMGB-1) [33,34]. SHK@HA-MPDA nanosystem induced CRT eversion and HMGB1 expression in the CT26 cells, but this process could be blocked by the addition of exogenous lactate (Fig. 5A, B, and C ). ...
Background:
There are few effective medications for treating colorectal cancer and liver metastases (CRLM). The interactions among glycolysis, epithelial-mesenchymal transition (EMT), and immune microenvironment contribute to the progression of CRLM. A main glycolytic enzyme pyruvate Kinase M2 (PKM2) is highly expressed in colorectal cancer and CRLM, and thus can be a potential therapeutic target.
Methods:
A therapeutic strategy was proposed and the shikonin-loaded and hyaluronic acid-modified MPDA nanoparticles (SHK@HA-MPDA) were designed for CRLM therapy via PKM2 inhibition for immunometabolic reprogramming. The treatment efficacy was evaluated in various murine models with liver metastasis of colorectal tumor.
Results:
SHK@HA-MPDA achieved tumor-targeted delivery via hyaluronic acid-mediated binding with the tumor-associated CD44, and efficiently arrested colorectal tumor growth. The inhibition of PKM2 by SHK@HA-MPDA led to the remodeling of the tumor immune microenvironment and reversing EMT by lactate abatement and the suppression of TGFβ signaling; the amount of cytotoxic effector CD8+ T cells was increased while the immunosuppressive MDSCs decreased.
Conclusion:
The work provided a promising targeted delivery strategy for CRLM treatment by regulating glycolysis, EMT, and anticancer immunity. An immunometabolic strategy for treating colorectal cancer liver metastases using the shikonin-loaded, hyaluronic acid-modified mesoporous polydopamine nanoparticles (SHK@HA-MPDA) via glycolysis inhibition, anticancer immunity activation, and EMT reversal. SHK@HA-MPDA can inhibit cytoplasmic PKM2 and glycolysis of the tumor and reduce lactate flux, and then activate the DCs and remodel the tumor immune microenvironment. The reduced lactate flux can reduce MDSC migration and suppress EMT.
... Li et al. (2020) reported that Astragalus polysaccharide inhibited breast cancer growth by regulating the immune response. The capacity of shikonin for activating anti-tumor immunity was investigated by Lin et al. (2015) Many of the previously published studies have paid attention to herbal medicines due to their low toxicity and high efficacy. Pseudobulbus Cremastrae seu Pleiones (PCSP), a pseudobulb of orchid plants is a typical TCM that has been used for clearing heat and detoxification, and for eliminating carbuncle and dispelling knots, etc. ...
Nanobiotechnology, the interface between biology and nanotechnology, has recently emerged in full bloom in the medical field due to its minimal side-effects and high efficiency. To broaden the application of nanobiotechnology, we composed gold nanoparticles from the extract of Pseudobulbus Cremastrae seu Pleiones (PCSP) using an efficient and green procedure. The biosynthesized Au nanoparticles containing PCSP (PCSP-AuNPs) were characterized by UV-vis spectroscopic, transmission electron microscopy (TEM), atomic force microscopy (AFM), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FT-IR), and Energy Dispersive X-ray (EDAX). After verifying the stability of PCSP-AuNPs, we detected its biosafety and immune-modulatory effects on RAW264.7 in vitro using NO assay, ELISA (TNF-α, IL-12p70, and IL-1β), and CCK-8 test. Furthermore, we examined the direct in vitro effects of PCSP-AuNPs on hepatocellular carcinomas (HCCs). Finally, we evaluated the immune regulation of PCSP-AuNPs using a mouse model with H22-tumor by testing the index of immune organs, splenic lymphocyte proliferation, cytokines levels (TNF-α and IL-10), and the CD4+/CD8+ cell ratio in the peripheral blood. Immunohistochemical analyses including H&E and PCNA staining were performed to investigate the anti-cancer efficacy and biocompatibility of PCSP-AuNPs. We found that PCSP-AuNPs not just possessed low toxicity, but also improved the immune-mediated antitumor response as compared to PCSP alone, suggesting its potential as a novel and efficient drug for liver cancer therapy.
... The latter, as a cancer vaccine system, often involves the effective employment of dendritic cells. Our laboratory in Academia Sinica, Taipei, performed a series of experimental studies [11][12][13][14], aiming to develop phytochemical-treated (PT) tumor cell lysate (TCL) and PT/TCL-pulsed (briefly co-cultured) and DC-based cancer vaccines for use against metastatic tumors. As seen in Figure 1, the experimental system makes good use of the iDC cultures. ...
... For instance, shikonin was shown to be highly effective as a key component of a cancer vaccine formulation for the treatment of tumor cell lysate (TCL)-pulsed, dendritic cell (DC)-based tumor vaccines [11]. Many of these activities were later shown to involve the DAMP signaling pathway [11][12][13] and/or the necroptotic autophagy activities [13]. ...
Plenty of evidence has recently shown that various inflammatory activities at the local tissue, organ, or even the whole body (systemic) level are strongly linked to many life-threatening chronic diseases, most notably various cancers. However, only very limited information is available for making good use of our supporting immune-modulatory therapeutics for the treatment of cancers. This may result from a lack of studies on specific remedies for efficacious control or modulatory suppression of inflammation-related cancerous diseases. Our group and laboratories were fortunate to have initiated and consistently pursued an integrated team-work program project, aimed at investigating selected medicinal herbs and the derived, purified phytochemical compounds. We focused on the study of key and specific immune-signaling mechanisms at the cellular and molecular levels. We were fortunate to obtain a series of fruitful research results. We believe that our key findings reported herein may be helpful for proposing future thematic and integrated research projects that aim to develop future phytochemical drugs against cancers. The mechanisms of the cellular and molecular systems involved in inflammation are becoming increasingly recognized as keystones for the development of future therapeutic approaches for many chronic and cancerous diseases. Recently, the immune checkpoint inhibitors such as antibodies against PD-1 and/or PD-L1 have been shown to be too expensive for general clinical use, and their effects far from optimal, often showing little or no effect or only short-term efficacy. These results point to the need for developing future immune-regulatory or modulatory therapeutics.
... 27 However, when transported out of a cell, HSP70 can also regulate immune function, including antigen presentation, dendritic cell maturation, NK cell, and MDSC activities. 28,29 Then this immunization resulted in a T cell-mediated immune response including a significant increase in CD4 þ or CD8 þ T cells, 29,30 even induction of cytokines, such as IL-1β and TNF-ɑ. 31 Our study also shows that the combination therapy of chemotherapy with RFH significantly enhanced the HSP 70 levels. ...
Background
To investigate the underlying molecular mechanisms of radiofrequency hyperthermia (RFH)-enhanced direct chemotherapy of pancreatic cancers.
Method
Rat ductal PaCa cell line DSL-6A/C1 and orthotopic pancreatic cancers of Lewis rats were divided into four study groups with various treatments: i) phosphate-buffered saline (PBS) as a control; ii) RFH alone; iii) intratumoral chemotherapy alone (gemcitabine); and (iv) combination therapy of gemcitabine plus intratumoral RFH at 42 °C for 30 min. In the in-vitro confirmation experiments, the viability and apoptosis of DSL-6A/C1 cells in each treatment group were evaluated using cell live/dead staining, flow cytometry, and Western blot. In the in vivo validation experiments, related proteins were evaluated by immunohistochemistry (IHC) staining of tumors.
Results
Of the in-vitro experiments, the lowest cell viability and more apoptotic cells were shown in the group with combination therapy compared to other treatments. Western blot data showed elevated Bax/Bcl-2, Caspase-3, and HSP70 expressions in DSL cells with combination therapy, compared to other treatments. Of the in vivo experiments, IHC staining detected the significantly increased expressions of HSP70, IL-1β, TNF-ɑ, Bax, and Caspase-3 in pancreatic cancer tissues of the animal group treated by combination therapy of gemcitabine with RFH.
Conclusion
Molecular imaging-guided interventional RFH can significantly enhance the chemotherapeutic effect on pancreatic cancers via potential molecular mechanisms of up-regulating Bax/caspase-3-dependent apoptosis pathways.
