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The anti-cancer components of Ganoderma lucidum possesses cardiovascular protective effect by regulating circular RNA expression

  • Guandong Laboratory Animal Monitoring Institute, China

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To examine the role of oral Ganoderma spore oil in cardiovascular disease, we used transverse aortic constriction (TAC) in mice to model pressure overload-induced cardiomyopathy. Our preliminary results demonstrated a potential cardioprotective role for spore oil extracted from Ganoderma. We found that Ganoderma treatment normalized ejection fraction and corrected the fractional shortening generated by TAC. We also found evidence of reduced left ventricular hypertrophy as assessed by left ventricular end diastolic diameter. Analysis of total RNA expression using cardiac tissue samples from these mice corroborated our findings. We found reduced expression of genes associated with heart failure, including a novel circular RNA circ-Foxo3. Thus our data provides evidence for Ganoderma lucidum as a potential cardioprotective agent, warranting further preclinical exploration.
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Oncoscience203 Oncoscience, Vol. 3 (7-8), July 2016
The anti-cancer components of Ganoderma lucidum possesses
cardiovascular protective effect by regulating circular RNA
Yi-Zhen Xie1,2,*, Fenghua Yang3,*, Weijiang Tan3, Xiangmin Li1,4, Chunwei Jiao2,
Ren Huang3, Burton B. Yang4,5
1 State Key Laboratory of Applied Microbiology Southern China, Guangdong Institute of Microbiology, Guangzhou, China
2 Guangdong Yuewei Edible Fungi Technology Co. Ltd., Guangzhou, China
3 Guangdong Laboratory Animals Monitoring Institute, Guangdong Provincial Key Laboratory of Laboratory Animals,
Guangzhou, Guangdong, China
4 Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, ON, Canada
5 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada
* These authors contributed equally
Correspondence to: Burton B. Yang, email:
Keywords: herbal medicine, Ganoderma lucidum, anti-cancer, heart function, circular RNA
Received: June 14, 2016 Accepted: August 12, 2016 Published: August 28, 2016
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original author and source are credited.
To examine the role of oral Ganoderma spore oil in cardiovascular disease, we
used transverse aortic constriction (TAC) in mice to model pressure overload-induced
cardiomyopathy. Our preliminary results demonstrated a potential cardioprotective
role for spore oil extracted from Ganoderma. We found that Ganoderma treatment
normalized ejection fraction and corrected the fractional shortening generated by
TAC. We also found evidence of reduced left ventricular hypertrophy as assessed
by left ventricular end diastolic diameter. Analysis of total RNA expression using
cardiac tissue samples from these mice corroborated our ndings. We found reduced
expression of genes associated with heart failure, including a novel circular RNA
circ-Foxo3. Thus our data provides evidence for Ganoderma lucidum as a potential
cardioprotective agent, warranting further preclinical exploration.
Complementary and alternative medicines have
attracted increasing attention as disease treatments,
adjuvants, and alternative supplements [1-4]. Medicinal
mushrooms comprise a large proportion of these
alternative products, among which Ganoderma lucidum
is the most highly studied [5-8]. Preclinical studies
have demonstrated anti-tumorigenic roles in a range
of medicinal mushrooms [9-11]. A Cochrane meta-
analysis showed that patients who had been administered
Ganoderma lucidum alongside chemo/radiotherapy
were more likely to respond positively compared to
chemo/radiotherapy alone. These trials demonstrated
improved immune functions as measured by increased
CD3, CD4, and CD8 immune response cells [12]. In
vitro, Ganoderma lucidum was also found to inhibit
proliferation and induce apoptosis in ovarian, colon, and
gastric cancer cell lines [13-15]. Ganoderma lucidum
contains beta glucans and other polysaccharides which
stimulate innate immunity and activate host dendritic cells
[16, 17]. Ganoderma lucidum also produces a group of
ganoderic acids, which have molecular structures that are
similar to steroids [18, 19].
An exploratory trial of 26 patients with hypertension
and/or dyslipidemia demonstrated that Ganoderma
lucidum treatment reduced total triglycerides and increased
HDL-cholesterol, implicating a cardio-protective role
[20]. We employed a transverse aortic constriction
(TAC) mouse model of pressure overload-induced
cardiomyopathy and heart failure to examine the role of
Ganoderma spore oil administration. The TAC model
induces an initial compensatory cardiac remodeling which
enhances cardiac contractility. Gradually, the response
to chronic overload leads to cardiac dilatation and heart
failure. The murine TAC model has been extensively used
to study cardiovascular disease and to elucidate signaling
pathways involved in cardiac hypertrophy and heart
TAC mice were administered oral Ganoderma spore
oil every other day for 14 days. The control group were
administered vegetable oil and an anti-hypertensive β2
adrenergic receptor antagonist. Mice were anesthetized
with 2% isourane inhalation for transthoracic
echocardiography and invasive hemodynamic assessment.
Transthoracic echocardiography was performed to
measure left ventricular ejection fraction (LVEF), left
ventricular fractional shortening (LVFS), left ventricular
end diastolic diameter, and cardiac output. Data analysis
was conducted in a blinded manner.
Relative to the 65.23% ejection fraction in
healthy sham mice, TAC mice were found to have a
43.26% ejection fraction (Fig 1A). This was below the
normal range of 55-75%, validating the TAC model
we employed. Treatment with the anti-hypertensive
medication approximated the normal range, while delivery
of Ganoderma spore oil recovered the stroke volume to
normal ranges. Consistent with these results, TAC mice
displayed 21.7% fractional shortening (Fig 1B). This
was within the mildly abnormal range of 20-25%, while
treatment with Ganoderma spore oil brought it to the
normal range of 25-45%. The TAC mice also showed
increased left ventricular end diastolic diameters, while
mice treated with Ganoderma spore oil did not exhibit the
same levels of left ventricular hypertrophy (Fig 2A). As a
result, mice treated with Ganoderma spore oil recovered to
physiologic cardiac output levels at 24.1 ml/min (Fig 2B).
This led to improved vascular perfusion within the mice
(Fig 2B). This suggests that Ganoderma spore oil increases
the heart function to meet the demands of the body.
After functional analyses, mice were sacriced and
heart tissue was harvested. Total RNA was extracted with
an RNeasy Mini Kit (Qiagen), followed by real-time PCR
measurement with miScriptSYBR GreenPCR Kit (Qiagen)
as described [21] to analyze levels of a circular RNA circ-
Fig 1. Ganoderma spore oil improves ejection fraction and fractional shortening. TAC mice showed decreased levels of LVEF (A)
and LVFS (B). Treatment with Ganoderma spore oil increased the levels of LVEF and LVFS.
Foxo3. We recently demonstrated that expression of circ-
Foxo3 RNA could inhibit tumor cell cycle progression
[22, 23] and promote cardiac senescence [24]. In this
study, mice injected with the chemotherapeutic agent
Doxorubicin (Dox) for induction of cardiomyopathy were
analyzed for RNA expression levels. Mice with reduced
cardiac function had increased expression of circ-Foxo3
RNA [24]. In the current study, we found that treatment
with Ganoderma spore oil decreased levels of circ-Foxo3
(Fig 3A).
To corroborate these results, we cultured mouse
cardiac broblasts and treated the cells with Ganoderma
spore oil, following hydrogen peroxide induced oxidative
stress. Relative to control groups, treatment with
Ganoderma spore oil decreased circ-Foxo3 levels in a
concentration- (Fig 3B) and time-dependent manner (Fig
Our previous studies have shown that the oil
fraction of Ganoderma spores could induce death in
versican-transformed cancer cells [25]. Further study
found that the Ganoderma spore oil could induce death
of cancer stem-like cells [6], potentially mediated by the
molecule ergosterol peroxide [19]. Our preclinical results
here show that Ganoderma spore oil has a protective role
within the cardiovascular system. Treatment in TAC mice
was found to normalize ejection fraction and correct the
fractional shortening generated by this model. We also
found evidence of reduced left ventricular hypertrophy
as assessed by left ventricular end diastolic diameter.
Thus in our TAC model, cardiac output was improved by
oral administration of Ganoderma spore oil. This data
provides rationale for further preclinical exploration of
Ganoderma lucidum as a cardioprotective agent.
This work was supported by the High-Level
Leading Talent Introduction Program of GDAS
(2016GDASRC-0102), The Introduction of Leading
Talent Project of Guangdong Province, and Open Fund of
the Guangdong Provincial Key Laboratory of Laboratory
Animals (No. 2013A061402001).
Fig 2. Ganoderma spore oil improves cardiac output. (A) TAC mice had increased left ventricular end diastolic diameters, while
treatment with Ganoderma spore oil decreased the end diastolic diameters. (B) Treatment with Ganoderma spore oil increased cardiac
No conicts of interest were disclosed.
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... Studies showed that G. lucidum spores or extracts of G. lucidum spores have protective capabilities such as retinal protection [74], cardiac protection [75][76][77], hepatic protection [78], intestinal protection [79], neuroprotective effect [80], bone marrow cells protection [81] and efficiency on apoptosis [74,79,82]. ...
... Moreover, treatment with G. lucidum spore oil (5mL, @P188/PEG400) nanosystem in mice significantly reduced X-ray-induced necrosis [75]. G. lucidum extracts also increased heart function [76,77]. ...
... Known since ancient times in traditional Chinese medicine as the "mushroom of immortality", G. lucidum has been used to promote well-being and longevity and is recognized for its hypoglycemic, immunomodulatory, antihypertensive, anti-diabetic, antioxidant, antihyperlipidaemic, antimutagenic, antiaging, antimicrobial, and hepatoprotective pharmacological properties [31,87]. In addition, it has also been widely used as an adjuvant in the treatment of various types of cancer [88,89], including breast cancer [90][91][92]. ...
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... Nonetheless, to our knowledge, there are no other additional available studies dealing with a similar pathology and methodology with which to compare. It is proved that medicinal mushrooms, such as G. lucidum, may improve EF in a rat model of doxorubicin-induced cardiomyopathy and TAC mice via anti-inflammatory and antiapoptotic mechanisms [35,36]. ...
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... Accordingly, Ganoderic acid A extracted from G. Lucidum was correlated to a hypoxic injury of the heart going through the PI3K/AKT pathway mediated rat H92 cardiomyocyte proliferation and apoptosis attenuation [320]. Furthermore, in the pressure-boosting irradiated cardiomyopathy mice model, one extract of spore oil was confirmed for the modification of cardiac function improvement through the circle RNA-FOXO3 axis, which is an important pathway associated with heart failure [321]. ...
... In a TAC-induced mouse model of cardiomyopathy induced by pressure overload, high circ Foxo3 expression was found to be involved in the protective mechanism of Ganoderma spore oil against cardiomyopathy [32]. In another study, we found that ganoderma spore oil improved cardiac function and reduced elevated circ-Foxo3 expression in doxorubicin (Dox) DOX-damaged hearts [33]. ...
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Most RNAs generated by the human genome have no protein-coding ability and are termed non-coding RNAs. Among these include circular RNAs, which include exonic circular RNAs (circRNA), mainly found in the cytoplasm, and intronic RNAs (ciRNA), predominantly detected in the nucleus. The biological functions of circular RNAs remain largely unknown, although ciRNAs have been reported to promote gene transcription, while circRNAs may function as microRNA sponges. We demonstrate that the circular RNA circ-Foxo3 was highly expressed in non-cancer cells and were associated with cell cycle progression. Silencing endogenous circ-Foxo3 promoted cell proliferation. Ectopic expression of circ-Foxo3 repressed cell cycle progression by binding to the cell cycle proteins cyclin-dependent kinase 2 (also known as cell division protein kinase 2 or CDK2) and cyclin-dependent kinase inhibitor 1 (or p21), resulting in the formation of a ternary complex. Normally, CDK2 interacts with cyclin A and cyclin E to facilitate cell cycle entry, while p21works to inhibit these interactions and arrest cell cycle progression. The formation of this circ-Foxo3-p21-CDK2 ternary complex arrested the function of CDK2 and blocked cell cycle progression.
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It has recently been shown that the upregulation of a pseudogene specific to a protein-coding gene could function as a sponge to bind multiple potential targeting microRNAs (miRNAs), resulting in increased gene expression. Similarly, it was recently demonstrated that circular RNAs can function as sponges for miRNAs, and could upregulate expression of mRNAs containing an identical sequence. Furthermore, some mRNAs are now known to not only translate protein, but also function to sponge miRNA binding, facilitating gene expression. Collectively, these appear to be effective mechanisms to ensure gene expression and protein activity. Here we show that expression of a member of the forkhead family of transcription factors, Foxo3, is regulated by the Foxo3 pseudogene (Foxo3P), and Foxo3 circular RNA, both of which bind to eight miRNAs. We found that the ectopic expression of the Foxo3P, Foxo3 circular RNA and Foxo3 mRNA could all suppress tumor growth and cancer cell proliferation and survival. Our results showed that at least three mechanisms are used to ensure protein translation of Foxo3, which reflects an essential role of Foxo3 and its corresponding non-coding RNAs.Oncogene advance online publication, 14 December 2015; doi:10.1038/onc.2015.460.
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Medicinal mushrooms in recent years have been the subject of many experiments searching for anticancer properties. We previously screened thirteen mushrooms for their potential in inhibiting tumor growth, and found that the water extract of Amauroderma rude exerted the highest activity. Previous studies have shown that the polysaccharides contained in the water extract were responsible for the anticancer properties. This study was designed to explore the potential effects of the polysaccharides on immune regulation and tumor growth. Using the crude Amauroderma rude extract, in vitro experiments showed that the capacities of spleen lymphocytes, macrophages, and natural killer cells were all increased. In vivo experiments showed that the extract increased macrophage metabolism, lymphocyte proliferation, and antibody production. In addition, the partially purified product stimulated the secretion of cytokines in vitro, and in vivo. Overall, the extract decreased tumor growth rates. Lastly, the active compound was purified and identified as polysaccharide F212. Most importantly, the purified polysaccharide had the highest activity in increasing lymphocyte proliferation. In summary, this molecule may serve as a lead compound for drug development.
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We have previously screened thirteen medicinal mushrooms for their potential anti-cancer activities in eleven different cell lines and found that the extract of Amauroderma rude exerted the highest capacity in inducing cancer cell death. The current study aimed to purify molecules mediating the anti-cancer cell activity. The extract of Amauroderma rude was subject to fractionation, silica gel chromatography, and HPLC. We purified a compound and identified it as ergosterol by EI-MS and NMR, which was expressed at the highest level in Amauroderma rude compared with other medicinal mushrooms tested. We found that ergosterol induced cancer cell death, which was time and concentration dependent. In the in vivo experiment, normal mice were injected with murine cancer cell line B16 that is very aggressive and caused mouse death severely. We found that treatment with ergosterol prolonged mouse survival. We found that ergosterol-mediated suppression of breast cancer cell viability occurred through apoptosis and that ergosterol up-regulated expression of the tumor suppressor Foxo3. In addition, the Foxo3 down-stream signaling molecules Fas, FasL, BimL, and BimS were up-regulated leading to apoptosis in human breast cancer cells MDA-MB-231. Our results suggest that ergosterol is the main anti-cancer ingredient in Amauroderma rude, which activated the apoptotic signal pathway. Ergosterol may serve as a potential lead for cancer therapy.
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All-trans retinoic acid (ATRA) is a differentiating agent for the treatment of acute promyelocytic leukemia (APL). However, the therapeutic efficacy of ATRA has limitations. Tetrandrine is a traditional Chinese medicinal herb extract with antitumor effects. In this study, we investigated the effects of tetrandrine on human PML-RARα-positive acute promyelocytic leukemia cells. Tetrandrine inhibited tumors in vivo. It induced autophagy and differentiation by triggering ROS generation and activating Notch1 signaling. Tetrandrine induced autophagy and differentiation in M5 type patient primary leukemia cells. The in vivo results indicated that low concentrations of tetrandrine inhibited leukemia cells proliferation and induced autophagy and then facilitated their differentiation, by activating ROS and Notch1 signaling. We suggest that tetrandrine is a potential agent for the treatment of APL by inducing differentiation of leukemia cells.
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Cordyceps species are known to produce numerous active components and are used for diverse medicinal purposes because of their varied physiological activities, including their ability to protect the liver from damage as well as their anticancer, antidepressant, anti-inflammatory, hypoglycemic, antimicrobial effects. Cordycepin, an adenosine derivative, differs from adenosine in that its ribose lacks an oxygen atom at the 3′ position. Several research groups have reported that cordycepin has antiviral activity against several viruses including influenza virus, plant viruses, human immunodeficiency virus(HIV), murine leukemia virus, and Epstein-Barr virus (EBV). In this study, we identify the epigenetic mechanisms by which cordycepin exerts its anti-gammaherpesvirus effects. We show that cordycepin possesses antitumor and antiviral activity against gastric carcinoma and EBV, respectively. A comparison of the CD50 values of cordycepin and its analogs showed that the lack of a 2′-hydroxyl group in cordycepin was critical for its relatively potent cytotoxicity. Cordycepin treatment decreased the rate of early apoptosis in SNU719 cells by up to 64%, but increased late apoptosis/necrosis by up to 31%. Interestingly, cordycepin increased BCL7A methylation in SNU719 cells by up to 58% and decreased demethylation by up to 37%. Consistent with these changes in methylation, cordycepin treatment significantly downregulated most EBV genes tested. Under the same conditions, cordycepin significantly decreased the frequency of Q and F promoter usage, and H3K4me3 histone enrichment was significantly reduced at several important EBV genomic loci. Extracellular and intracellular EBV genome copy numbers were reduced by up to 55% and 30%, respectively, in response to 125 μM cordycepin treatment. Finally, cordycepin significantly suppressed the transfer of EBV from LCL-EBV-GFP to AGS cells, indicating that EBV infection of gastric epithelial cells was inhibited. These results suggest that cordycepin has antiviral and antitumor activities against gammaherpesviruses and host cells latently infected with virus.
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Melanoma is one of the fastest-rising types of cancer in North American. Accumulating evidence suggests that anti-tumor immune tolerance plays a critical role in tumor development. B16 melanoma cells were injected into wild type and miR-17 overexpressing transgenic mice. Tumor growth was monitored and tumor bearing mice were sacrificed by the end of the forth week. Peripheral blood and spleen cells were subject to flow cytometry analysis and tumor samples were subject to immunohistochemistry staining. Meanwhile, Jurkat cells transfected with mock-control or miR-17 overexpressing plasmid were co-cultured with B16 cells. The influence of miR-17 on cell cycle, proliferation and survival was evaluated. The melanoma tumors formed in mice overexpressing miR-17 were less than that in wild type mice. In addition, the miR-17 tumors were less invasive and less angiogenic. The percentage of CD8+ T cells was suppressed in miR-17 transgenic mice before melanoma cell injection. Its level was significantly increased upon tumor grafting. More tumor infiltrating CD8+ cytotoxic T lymphocyte could be found in transgenic mice with tumor formation. Luciferase assay and protein analysis indicated that STAT3 was the target of miR-17. Decreased levels of STAT3 were associated with miR-17 over-expression. Down-regulation of STAT3 in Jurkat cells promoted cell proliferation and mitosis. MiR-17 inhibits melanoma growth by stimulating CD8+ T cells mediated host immune response, which is due to its regulation of STAT3.
Chemotherapy is one of the most common therapeutic option for metastatic tumors and hematological malignancies. ABCB1-mediated multidrug resistance is the major obstacle for chemotherapy. Natural products with diversified structures are ideal source of ABCB1 modulators. Ganoderenic acid B, a lanostane-type triterpene isolated from Ganoderma lucidum, exhibited potent reversal effect on ABCB1-mediated multidrug resistance of HepG2/ADM cells to doxorubicin, vincristine and paclitaxel. Similarly, ganoderenic acid B could also significantly reverse the resistance of ABCB1-overexpressing MCF-7/ADR cells to doxorubicin. Furthermore, ganoderenic acid B notably enhanced intracellular accumulation of rhodamine-123 in HepG2/ADM cells through inhibition of its efflux. ABCB1 siRNA interference assay indicated that the reversal activity of ganoderenic acid B was dependent on ABCB1. Further mechanistic investigations found that ganoderenic acid B did not alter the expression level of ABCB1 and the activity of ABCB1 ATPase. Molecular docking model displayed that the positions of ganoderenic acid B binding to ABCB1 were different from the region of verapamil interacted with ABCB1. Collectively, ganoderenic acid B can enhance the cytotoxicity of chemotherapeutics towards ABCB1-mediated MDR cancer cells via inhibition of the transport function of ABCB1. These findings provide evidence that ganoderenic acid B has the potential to be developed into an ABCB1-mediated multidrug resistance reversal agent.