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Conquering triple-negative breast cancer

2020-09-24
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According to the 2018 International Agency for Research on Cancer (IARC) survey data, the incidence of breast cancer in female cancers worldwide is 24.2%, ranking first among female cancers, of which 52.9% occur in developing countries. From the age of onset, the incidence of breast cancer gradually rises after the age of 20, reaching a peak between the ages of 45 and 50.
Triple negative breast cancer (TNBC) is one of all breast cancers. It is called “three negatives” because the three main therapeutic targets of estrogen receptor, progesterone receptor and HER-2 are negative. About 15% of all breast cancers. This type of breast cancer is considered to be the “most toxic and dangerous” breast cancer due to the lack of targets and the high risk of recurrence and metastasis. Among them, triple-negative breast cancer, which is still progressing after many rounds of treatment, is even closer to “exhaustion.” The proportion of patients with tumor shrinkage after conventional treatment is less than 10%.
Nature is full of compounds that are harmful to us, but medical scientists have been showing us how to use them to benefit mankind and even become life-saving drugs. Recently, a breakthrough discovery by Australian scientists is the latest example. The team demonstrated how an ingredient in bee venom can be used as a “very powerful” weapon against breast cancer. This research was conducted at the Harry Perkins Institute of Medical Research in Australia. We have seen how certain peptides and proteins in bee venom are used to transport drugs across the blood-brain barrier, but Dr. Duffy’s research focuses on how It is used to treat various breast cancers. Her research focuses on an active compound called melittin in bee venom and how it induces cell death in different clinical subtypes of breast cancer. The research was published in the journal of Nature Precision Oncology.

Nature Precision Oncology
Source: npj Precision Oncology

Bee venom and melittin can specifically reduce the viability of breast cancer cells

breast cancer cells

Source: npj Precision Oncology

In order to evaluate the anti-cancer efficacy and selectivity, the researchers evaluated bee venom collected from Australia. The venom showed high anti-cancer selectivity, with higher efficacy in TNBC and HER2-rich breast cancer cell lines, followed by They are intraluminal breast cancer cells and have the least impact on normal cells.
The researchers developed a mouse monoclonal antibody that recognizes melittin to evaluate the relative abundance of melittin in all honeybee and bumblebee venom samples by ELISA. According to the above activity study, the relative abundance of melittin is not significantly different in all bee venom samples at different locations (two-way analysis of variance, p>0.999). However, the concentration of melittin in the honeybee samples was significantly higher compared to the bumblebee venom and isotype IgG controls.
The anti-cancer effect of melittin was confirmed by in vitro blocking experiments, in which the researchers used anti-mellittin antibodies to rescue the cell viability of HDFa and SUM159 cells. These data indicate that the melittin present in bee venom is the most prominent bioactive anticancer compound in the venom studied.
Bee venom and melittin can induce breast cancer cell death
In order to find out the mechanism of cell death, TNBC cells were treated with bee venom or melittin IC50 for 18 and 24 hours, respectively, and treated with lysed caspase-3 to quantify the death of apoptotic cells. Western blotting confirmed the induction of caspase-3 lysed in SUM159 cells. Melittin alone can induce higher levels of cancer cell apoptosis than bee venom at 18 and 24 hours after treatment.

Bee venom and melittin can induce breast cancer cell death

induce death of breast cancer cells

Source: npj Precision Oncology

In order to quantify the apoptotic, necrotic, or dead cell population after treatment, the researchers performed the annexin V-FITC cell apoptosis detection analysis. SUM159 cells were exposed to vehicle, bee venom or melittin using IC50 concentration, and processed by flow cytometry after 60 minutes of treatment. The researchers found that compared with bee venom (8.3±1.9%) and vehicle control (4.8±0.4%), samples treated with melittin (23.6±5.7%) had significantly more late apoptotic or necrotic cells (23.6±5.7) %), but there was no significant difference in the levels of early apoptosis or necrotic cells. In order to characterize the kinetics of cell death in a short period of time, the cell viability of HDFa, SKBR3 and SUM159 cells treated with melittin at IC50 concentration for up to 1 hour was measured. Bee venom will rapidly reduce cell viability, and there is no significant difference between normal cells and cancer cell lines within one hour. On the contrary, from 10 minutes, melittin significantly reduced the viability of the two breast cancer cells compared with normal cells, and from 30 minutes, SUM159 was significantly higher than SKBR3 (two-way ANOVA, p <0.0001). Confocal microscopy and scanning electron microscopy of live cells in SKBR3 and SUM159 cells indicate that the plasma membrane will quickly rupture and shrink within 10 to 60 minutes when treated with melittin and melittin.

Bee venom and melittin inhibit RTK phosphorylation

inhibit RTK phosphorylation

Source: npj Precision Oncology

In SUM159, bee venom and melittin significantly down-regulate p-EGFR within 10 to 20 minutes. SUM159 cells trigger ERK signals due to negative regulatory feedback loops in MAPK and Akt pathways to protect cells from the release of apoptotic cell death. The anti-melittin antibody showed that the content of melittin in the lysate of both cell lines increased over time, and the signal of melittin treatment in the two cell lines was stronger compared with bee venom. To characterize the effect on signaling pathways in another TNBC model, the researchers performed western blot analysis on MDA-MB-231 cells, in which EGF treatment phosphorylates EGFR and induces EGFR expression. Unlike SUM159 cells, the stimulation of EGFR by EGF is not related to the increase of p-Akt phosphorylation, which may be due to the separation between EGFR signaling and Akt pathway.
Considering that breast cancer cells rich in TNBC and HER2 are highly dependent on the activation of EGFR and HER2, the researchers conducted a bioluminescence resonance energy transfer (BRET) experiment to determine whether melittin interferes with the binding of EGF and EGFR. The BRET signal of TAMRA-EGF and FITC-DEDE-melittin increased in a dose-dependent manner, while the amplitude of FITC-melittin was smaller. At the same concentration, the BRET ratio of FITC-DEDE-melittin is much higher than that of FITC-melittin, and the maximum BRET ratio can be reached quickly at each dose. In order to determine the specificity of melittin binding to EGFR at the EGF binding site, saturated BRET analysis was performed to evaluate the competition of EGF with each NanoLuc-EGFR-binding peptide. When 1 µM EGF is present, the binding of TAMRA-EGF to NanoLuc-EGFR is saturated and significantly reduced, while the BRET signals of FITC melittin and FITC-DEDE-melittin are not saturated, and in the presence or absence of 1 µM EGF There is no significant difference in the following, which indicates that neither melittin nor DEDE-melittin binds to the EGF binding site.
In short, melittin is integrated into the plasma membrane of cancer cells through the charged sequence at the C-terminus, thereby inducing plasma membrane remodeling and destruction. The BRET data showed that melittin can be located within 10 nm from the RTK without interfering with the binding sites of endogenous growth factors.

Results and discussion

Studies have shown that bee venom and melittin can inhibit ligand-induced phosphorylation of EGFR and HER2, thereby dynamically regulating downstream signaling pathways in breast cancer cells. Melittin can also enter cells to directly or indirectly regulate downstream signal transduction pathways. The selectivity of melittin for HER2-driven tumors also provides a further case for its combined use with HER2 targeted drugs. The membrane destruction properties of melittin can enhance the intrinsic dynamics of its cytotoxic payload.
The researchers also revealed new opportunities to modify specific regions of melittin to further improve the efficacy and targeting specificity of malignant cells. Engineered targeting peptides, such as RGD1-melittin, can be delivered intravenously to achieve more selective homing and uptake into tumor cells. However, before human trials, further studies on the toxicity and maximum tolerated dose of these peptides will be required.
Melittin can be used on a global scale and provides cost-effective and easy-to-use treatment options in some remote or underdeveloped areas. At present, further research is needed to evaluate whether the venom of certain genotypes of bees has stronger or specific anti-cancer activity and can be used. Overall, the results of this study can be used to assist in the development of new treatments for multiple cancers associated with frequent drug resistance and poor prognosis. Medicilon, as a new drug research and development CRO, will continue to pay attention to this research progress, hoping to help breast cancer new drug research and development.

About Medicilon

Medicilon (stock code: 688202) was established in 2004 and is headquartered in Shanghai. It is committed to providing a full range of pre-clinical new drug research services for global pharmaceutical companies, research institutions and scientific researchers. Medicilon’s one-stop integrated service helps customers accelerate the development of new drugs with strong project management and more efficient and cost-effective R&D services. The services cover the entire process of preclinical new drug research in medicine, including drug discovery, pharmaceutical research and clinical trials. Pre-research. Medicilon grows together with high-quality customers at home and abroad, and provides new drug research and development services to more than 700 customers around the world. Medicilon will continue to base itself on a global perspective, gather Chinese innovation, and contribute to human health!

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