Advances in the research of plant-derived natural products against retinoblastoma

INTRODUCTION

Retinoblastoma (RB) is the most common primary intraocular malignancy in childhood

, with more than 90% of cases presenting before the age of 5 years

. It is mainly due to the loss of the double allele of the

gene in the developing retina

, as well as the appearance of other genetic and epigenetic alterations eventually leading to the development of cancer

. The combination of systemic and local chemotherapy, local condensation, laser therapy, radiotherapy and surgery has significantly improved the survival rate of children.Treatment failure in some children because of the toxic side effects and drug resistance to chemotherapy, increased risk of second primary tumor development with radiotherapy, and the ease of recurrence with local treatment

has placed a huge burden on families and society, and new therapeutic agents need to be sought to address the problem.

王爺平素不這樣，像一塊生冷的鐵；實在是被氣著了。中午從他家往車上裝那些木偶時，一個搬東西的人毛手毛腳，把鐵架子一角直直壓在了一個木偶身上，心疼得王爺一個大跨步上去，把那鐵架子嘩啦一下就推開了，倒叫旁邊的人著實一驚：他幾乎是歪著身子跨上前去的，若不小心摔著，83歲的人了怎能經得起。

沒錯，安安姓凌，其實是凌薇同父異母的妹妹。是凌宇生二十多年前與酒店小姐一夜風流生的孩子。凌宇生的老婆，也就是凌薇的母親是個十足的悍婦，所以二十年來，凌宇生只敢給安安母女剛夠維持生活的錢，為了安安讀書，安安母親后來又下海當了好幾年小姐，直到人老珠黃。

Natural products from plants, animals, microorganisms and minerals have long been a source of drugs for the treatment of human diseases

. And studies have confirmed that such products can be used as potential anti-cancer drugs or adjuvants for chemotherapy

, which are valuable for both cancer prevention and treatment and play an important role in the exploration and development of new anti-cancer drugs.More than 60% of antitumor drugs show a close association with natural products

according to the literature, where natural products of plant origin are the main source of anticancer drugs. About 3/4 of the current drugs for cancer treatment are derived from plants

, such as paclitaxel,camptothecin, etoposide and vincristine have been approved for the clinical treatment of many cancers

, providing more options for attacking cancer. This class of natural products has been widely used in the research of anti-RB therapy, which can produce anti-RB effects by inducing RB cell apoptosis,inhibiting RB cell invasion, triggering the RB cell cycle arrest,and regulating RB cell resistance in various ways.

PROMOTE RETINOBLASTOMA CELL APOPTOSIS

Apoptosis is the main mechanism regulating cell death, but apoptosis of tumor cells are being often in a dysregulated state in human cancer

. The study of the molecular mechanisms of apoptosis in cancer cells to obtain effective drugs that promote apoptosis in tumor cells is widely used in the current strategies for anticancer drug development and is a key direction of antitumor research.

Phosphatidyl inositol 3-kinase/serine threonine protein kinase (PI3K/AKT) is one of the most important intracellular signaling pathways, which can be activated by insulin, growth factors and cytokines under physiological conditions. This pathway plays an important role in glucose metabolism, synthesis of macromolecules and maintenance of tissue redox homeostasis

, supporting the metabolic homeostasis of the system and promoting the growth and metabolism of individual cells

. The PI3K/AKT signaling pathway exhibit frequent mutations and enhanced activity in cancer

, and activation of the pathway induces changes in the expression of a variety of downstream genes that promote tumorigenesis and progression, and has been found to be abnormal in almost all human cancers

.

A variety of plant-derived natural active ingredients have shown good effects in inducing RB cell apoptosis

in the current study. These active ingredients exert anti-RB effects by modulating the activity of different signaling pathways in RB cells, altering the expression of downstream related genes,and inducing an increase in intracellular apoptotic signals.

Invasion and migration is an important hallmark of malignant tumor progression, which involves various factors such as degradation of extracellular matrix, epithelial-mesenchymal transition, tumor angiogenesis, and the development of inflammatory tumor microenvironment

, and is closely related to poor prognosis of cancer. Inhibition of tumor metastasis is important for the treatment of tumors, which can buy time for the treatment of tumor patients and improve their survival and cure rate.

Tumor protein 53(p53) is an important tumor suppressor gene involved in the regulation of various cellular activities such as apoptosis of tumor cells, genomic stability, tumor angiogenesis, metabolism of cancer cells and tumor microenvironment, and has a role in the prevention of tumorigenesis

. The

gene was found to be one of the most frequently mutated genes in tumor cells,and inactivation of this gene is a key driver in the development of several cancers

, and mutations in the p53 protein are present in approximately half of human tumors, and alterations in the components of the p53-mediated tumor suppressor pathway also occur in the other half

. So regulation of the p53 signaling pathway is an important antitumor strategy.Ziyuglycoside I is one of the main active ingredients isolated from the traditional Chinese herb Diel root. Zhu

found that Ziyuglycoside I significantly inhibited the viability of WERI-RB1 cells. The cellular protein assay after the action of Ziyuglycoside I found that Ziyuglycoside I upregulated Bcl-2 and Mito- by inducing the activity of phosphorylated and acetylated p53 proteins in the cells.Cyto c protein expression decreased, while inducing Bax and Cyto-cyto c protein expression in cells, which promoted apoptosis. Inhibition of WERI-RB1 cells by Ziyuglycoside I was significantly decreased after blocking p53 indicating that the p53 pathway plays an important role in Ziyuglycoside I-induced apoptosis of WERI-RB1 cells.

2.優秀戲曲藝術家。隨著桂林戲曲的不斷發展，形成了一批為優秀的戲劇藝術家、演員及曲藝傳承人，如桂劇藝術家秦彩霞、周小蘭魁、羅桂霞、張樹萍、曾定國、張浩和周子瑜等，彩調藝術家傅錦華、唐玉剛、秦蘭香和林葉梅，文場代傳承人何紅玉、陳秀芬和李偉，漁鼓藝術家李蔚琛、秦小桔和彭承紅等。

The mitogen-activated protein kinase (MAPK) family is a group of highly conserved protein kinases

that

can co-regulate extracellular signal-regulated kinase (ERK). Activation of the MAPK/ERK pathway can significantly increase the expression of glucose transporters and a large number of glycolytic genes, resulting in altered cellular metabolism and promoting tumor growth and proliferation

. Therefore, blockade of MAPK/ERK signaling pathway can inhibit tumor growth, increase tumor cell apoptosis, and exert anti-tumor effects. Puerarin, a naturally active isoflavone extracted from

, was found by Yang

to significantly inhibit the proliferation of RB Y79 cells and SO-RB50 cells in a dose- and time-dependent manner by MTT assay. Protein blotting assay revealed that p-MEK and p-ERK protein expression in RB cells treated with Puerarin was significantly lower than that in the underage group, and the inhibitory effect of Puerarin on RB cells could be counteracted with MAPK/ERK agonists, confirming that Puerarin exerts its anti-RB effect by inhibiting the MAPK/ERK pathway.Both p38 mitogen-activated protein kinase (p38 MAPK)and c-Jun amino-terminal kinase terminal kinase (JNK) are members of the MAPK family

. Yu

found that the p38 MAPK and JNK pathways play an important role in Y79 cell apoptosis induced by the plant polyphenol compound curcumin, and curcumin can promote apoptosis of Y79 cells by activating p38 MAP and JNK in cells and upregulating caspase-9 and caspase-3 activities, while the induction of caspase-9 and caspase-3 protein expression by curcumin was significantly decreased after the use of p38 MAPK or JNK inhibitors, along with a decrease in the apoptosis rate of Y79 cells, thus showing that curcumin produces inhibition of Y79 cells through the p38 MAPK and JNK pathways. Quercetin is a flavonol found in many vegetables. Liu and Zhou

found that quercetin induced apoptosis in Y79 cells by a mechanism similar to that curcumin, also by activating the JNK and p38 MAPK pathways in cells to enhance the activity of caspase-9 and caspase-3, producing an anti-Y79 cellular effect, and this inhibition could also be reversed by JNK inhibitors and p38 MAPK inhibitors.

The four Janus kinases(JAK1, JAK2, JAK3, TYK2) and seven signal transduction and activator of transcription

(STAT1, STAT2, STAT3,STAT4, STAT5A, STAT5B, STAT6) together constitute the JAK-STAT signaling pathway

, which is essential for the signaling of various metabolic-related hormones and cytokines in the cells

. The JAK/STAT signaling pathway plays a variety of important biological functions in malignant cells

,and this pathway mediates almost all immunoregulatory processes, including tumor cell recognition and tumor immune escape

, which are closely related to tumor progression and prognosis. Studies have confirmed that JAK-1 and STAT-3 protein expression levels are independent risk factors for patient prognosis in patients with colon cancer

, and JAK/STAT was also found to be closely associated with the malignant biological behavior of tumors in a study of RB. Curcumin extracted from turmeric has been shown to have antitumor activity in recent years. Li

used curcumin to act on RB SO-RB50 cells and Y79 cells and found that curcumin significantly inhibited the proliferation, colony formation,migration and invasion of SO-RB50 cells and Y79 cells,while promoting both SO-RB50 and Y79 RB cells apoptosis.Upregulation of miR-99a was detected in curcumin-treated cells, and the phosphorylation levels of JAK1, STAT1 and STAT3 proteins were inhibited. The inhibitory effect of curcumin on the JAK/STAT pathway disappeared after knockdown of miR-99a. It is evident that curcumin can exert anti-SO-RB50 cells and Y79 cells by inactivating the JAK/STAT signaling pathway through the regulation of miR-99a.

由上述步驟可知，整個項目在施工階段和運營階段均面臨極大風險隱患。在采取風險控制措施后，項目的風險可以降低到可接受的等級。

XPC基因單核苷酸多態性與肺癌相關性的研究進展…………………………………………………… 范曉凡等（14）：2007

p-AKT/AKT ratios were also significantly lower than in the control group, while the inhibitory effect of CGA on HXORB44 cells could be reversed after the use of PI3K activator,confirming that CGA has the ability to inhibit the PI3K/AKT pathway in HXO-RB44 cells effect, and that CGA promoted apoptosis in HXO-RB44 cells through the inhibition of this pathway.

Additional results of anti-RB studies using active ingredients such as Neferine and Sinomenine

showed that both can produce clear anti-RB cell invasion through different pathways.The above studies indicate that there are a large number of active ingredients in plants that can inhibit tumor metastasis,and their study can help to develop drugs or adjuvant classes of drugs that inhibit tumor metastasis, solve the most difficult problems in tumor treatment, and improve patient survival.

In addition, extracts derived from plants can also exert antitumor effects on RB through signaling pathways such as PERK-ATF4-CHOP pathway and miR-937/FOXQ1 pathway

. The above studies indicate that there are abnormalities in the regulation of a large number of signaling pathways in RB cells, and the study of signaling pathways is an important direction for the development of antitumor drugs. Since this class of active ingredient drugs can produce antitumor effects on RB in different pathways, they have great potential in the treatment of diseases.

INHIBITION OF RETINOBLASTOMA CELL INVASION AND MIGRATION

Matrine is an alkaloid isolated from the plant herb bitter ginseng

. Zhang

used bittersweet to act on RB Y79 lineage cells, they found that apoptosis of Y79 cells increased and cell proliferation was significantly inhibited, and the mRNA expression of PI3K and AKT in the cells was significantly inhibited by assay analysis, and this inhibition was closely related to the concentration of bittersweet, while the protein expression of PI3K and AKT in the cells also showed a dose-dependent decrease. The anti-Y79 cellular effect of matrine was also confirmed in a study by Chen and Ai

The expression of p85α subunit protein of PI3K and AKT protein was significantly decreased in Y79 cells treated with matrine, leading to a decrease in the expression of its downstream apoptosis inhibitory protein Bcl-2 protein and a significant increase in the expression of the pro-apoptotic Bax protein, which induced apoptosis in Y79 cells. Liu

used Eucommia chlorogenic acid (CGA), a natural product extracted from

, to treat RB HXO-RB44 cells and found that apoptosis of HXO-RB44 cells was significantly increased after treatment with CGA, and analysis of proteins in the cells revealed that the drug group cells had Ki67, NQO1,TrxR, p-PI3K, and Nrf2 expression were significantly lower in the drug group cells than in the control group, and both

Nuclear transcription factor-κB (NF-κB) is an important transcription factor in biological cells and has an important role in the immune system in regulating the expression of a variety of cellular response genes

. A growing number of studies are now confirming that inflammation is closely related to the generation and development of human cancers,and NF-κB, as a key molecule linking persistent infection and chronic inflammation to increased cancer risk

, is a current hot topic in antitumor research. Mu

found that the proliferation and migration ability of cells were significantly decreased after the action of curcumin on WERI-RB1 cells,and the protein expression assay of the cells revealed that the p65 nuclear translocation was significantly inhibited and the protein expression of NF-κB downstream factors matrix metalloproteinase (MMP)-2, MMP-9, and vascular endothelial growth factor (VEGF) was significantly decreased, indicating that the inhibitory effect of curcumin on WERI- RB1 cells was achieved by regulating the NF-κB signaling pathway. Elemene is a natural component with anticancer effects extracted from the ginger family, and Wei

found that the proteins of NF-κB and the downstream factor surviving were highly expressed in human RB HXO-RB44 cells, and the expression of both proteins decreased significantly after treatment of HXO-RB44 cells with elemene, indicating that elemene may promote apoptosis in HXO-RB44 cells by inhibiting NF-κB activity, downregulating the expression of Survivin gene and thus promoting apoptosis in HXO-RB44 cells. Thus, in RB,natural active ingredients of plant origin may inhibit tumor progression by down-regulating NF-κB activity and thereby inhibiting tumor progression.

MMPs are key cytokines in the process of cell invasion, which can make tumor cells metastasize easily by degrading the extracellular matrix in tissues, and are a popular target in the current research of antitumor drugs. The results of Li

showed that Astragalus polysaccharide extracted from the root meridian of

could significantly reduce the number of RB44 cells penetrating the basement membrane of Transwell chambers, and the detection of MMPs in the cells treated with Astragalus polysaccharide revealed that the expression of MMP-9 and MMP-2 proteins in the cells was significantly down-regulated, and the degradation ability of extracellular matrix was inhibited, leading to a decrease in cell invasiveness.

Epithelial to mesenchymal transition (EMT) is a biological process in which epithelial cells are transformed into cells with a mesenchymal phenotype through a specific program

and has an important role in tumor metastasis. in which transforming growth factor-β1 (TGF-β1) is the main inducer of tumorigenesis EMT and can play an important role by TGF-β1 is the main inducer of tumorigenic EMT, and can exert EMT effects by regulating small molecule proteins such as N-calmodulin, waveform protein and E-calmodulin.In the process of EMT, the polarized form of epithelial cells is destroyed and gradually transformed into mesenchymal cells, which makes the invasiveness and metastasis of tumor significantly enhanced. Therefore, the inhibition of the EMT process of tumor cells can effectively reduce the chance of tumor metastasis. Pinocembrin is a flavonoid extracted from plants such as nut pine, eucalyptus leaves and acacia gum,and Chen

used Pinocembrin in an experimental study of the invasiveness of Y79 cells and found that the invasive migratory capacity of Y79 cells was reduced after the use of Pinocembrin, and the detection of relevant proteins in the cells revealed that Pinocembrin caused the N-calmodulin, αv and β3 integrins and wave proteins in Y79 cells to expression significantly, resulting in a blocked TGF-β1-induced EMT process in Y79 cells. Berberine is an alkaloid extracted from the buttercup plant Huanglian, and in an anti-RB study using Berberine, Wang

found that it inhibited PI3K/AKT and p38 pathways and decreased E-cadherin protein amounts,while upregulating the expression of waveform proteins and α-SMA, resulting in a decrease in the invasion and migration of RB cells.

Angiogenesis is an important event in the process of tumor growth and hematogenous metastasis

, where tumor cells induce the sprouting and generation of new capillaries by secreting high levels of pro-angiogenic factors to form an abnormal vascular network. The newly formed blood vessels provide oxygen and nutrients to tumor tissues and provide channels for their distant metastasis, which is essential for tumor growth and spread

.At the same time, the immaturity and high permeability of newly formed vessels lead to a hypoxic microenvironment in parts of the tumor due to poor perfusion, and this microenvironment can promote the invasion of tumor cells,enhance the anti-immune response of tumors, and increase the drug resistance of tumor cells

. Therefore, conducting research on anti-treatment angiogenic drugs will help to improve the efficacy of clinical anti-tumor therapy. VEGF and its receptor (VEGFR) are the key regulators identified to promote tumor angiogenesis, and previous studies have demonstrated that inhibition of their expression can effectively reduce tumor angiogenesis. Fisetin is a small molecule compound extracted from plants of the

family,and Wang

found that the pro-angiogenic ability of Fisetin-treated Y79 cells was decreased, and further studies revealed that both VEGFR protein and mRNA expression in the cells were significantly inhibited, and there was a dosedependent inhibition of VEGFR expression and the ability to induce angiogenesis in Y79 cells at different concentrations of Fisetin. Quercetin (Que) is a flavonoid with antitumor activity and is widely found in plant-based herbs. Song

found that Que inhibited the proliferation of RB cells and also inhibited the VEGFR expression in the cells, blocking the angiogenesis-promoting effect of RB cells, and observed that Que inhibited RB cell-induced angiogenesis. The effect of Que on RB cell-induced angiogenesis was also observed to be dose-dependent. In addition, Wang

found that methyl lotusine from lotus seed germ could also significantly inhibit RB cell-induced microangiogenesis. These experimental studies indicate that plant activities with anti-RB angiogenic effects are widely available, and the results of the experiments provide a basis for further drug development and show that active ingredients derived from plants have great potential in the therapeutic field of anti-RB angiogenesis.

人們對攝影技藝趣味化的追求正體現出一種對自由想象、表現與創作的追求藝術。拍攝或制作時采用一些特殊技法，如拍攝時采用慢速快門、多次曝光；制作時采用加網、局部虛化、影畫結合、浮雕、多底合成、影調（黑白）及色調（彩色）分離、多影合成、套影、景像合成等特殊效果的制作使照片呈現特殊的畫面效果。