TWI549677B - Use of ugonin compounds in manufacturing drugs for treating skin cancer - Google Patents

Description

Use of Ugonin compound for preparing medicine for treating skin cancer

The present invention relates to the use of a Ugonin compound for the preparation of a medicament for treating skin cancer, in particular to the ability of a Ugonin compound to promote skin cancer cell apoptosis (apoptosis) and inhibit cancer cell migration and invasion. The compound is low in normal cytotoxicity and thus can be developed as a medicine for treating skin cancer with specificity.

Skin cancer is mainly divided into two types: melanoma skin cancers (MSC) and non-melanoma skin cancers (NMSC). Non-melanocyte cancer includes: basal cell carcinomas (BCCs), which have the highest incidence but the lowest malignancy, account for about 40% of the incidence of skin cancer; squamous cell carcinoma is the most common incidence and malignancy ( Squamous cell carcinomas (SCCs), which together account for more than 75% of skin cancer. The lowest incidence but the highest malignancy is melanoma, which accounts for about 10% of the incidence of skin cancer.

Traditionally, in the treatment of skin cancer, surgical treatment is the most common treatment. Also included are: local chemotherapy, cryotherapy, photodynamic therapy (PDT), and adiotherapy. Local chemotherapy drugs such as 5-fluouracil (5-FU) have been used to treat cancer cell expansion; other chemotherapy drugs like Methotrexate, 5-mercaptouracil, Nitrogened mustard, Actinomycin D, Intravenous cisplatin, Intralesional bleomycin or Imiquimod, although therapeutically effective, can cause considerable side effects such as pain, ulcers, scarring, pigmentation or loss, and inflammatory reactions. Furthermore, traditional methods of treating cancer, whether Local treatment or systemic treatment, in addition to killing cancer cells, also causes damage to normal cells, or with a variety of side effects, which greatly affects the patient's health and increases the physiological and psychological burden of the patient. Therefore, how to propose a better anti-skin cancer product is a development focus.

Helminthostachys zeylanica (L) Hook. belongs to Ophioglossacrae, Helminthostachyaceae, commonly known as: inverted unicorn, valerian, nailed mantle, Taiwan, Southeast Asia and India. Anti-inflammatory herbs commonly used in other regions, whose roots are dried and decoction, have the effects of relieving heat, promoting blood circulation, reducing inflammation, relieving pain, and producing muscle. They can also be applied to internal pain, tuberculosis and poisonous snake wounds. In terms of phytochemical composition, current studies have shown that compounds obtained by extracting and purifying the rhizome of C. sylvestris include: flavonoids, new flavonoids with isoprene skeleton (Ugonin A~Ugonin T), New terpenoids (Ugonstilbene A~Ugonstilbene C) and quercetin. From the results of the study, it was found that the extract of Ceylon sylvestris has excellent anti-oxidation, anti-inflammatory, protective cell and anti-cell proliferation activities. In addition, Ugonin K, a component of Ceylon, has promoted osteogenic cell maturation and induced oxygenation by reactive oxygen species. The protective effect of cell death. However, the use of Ugonin compounds extracted and purified from C. sylvestris has not been reported for anti-cancer, and remains to be studied in the development of skin cancer.

Nowadays, the inventor is that in view of the above-mentioned existing drugs for treating skin cancer, there are still many defects in the actual implementation, so it is a tireless spirit, and with its rich professional knowledge and years of practical experience, Improvements have been made and the present invention has been developed based on this.

The main object of the present invention is to provide a Ugonin compound which promotes apoptosis of skin cancer cells and inhibits migration and invasion of cancer cells. This Ugonin compound has low toxicity to normal cells, and thus can be developed as a medicine for treating skin cancer with specificity.

In order to achieve the above-mentioned object, the Ugonin compound of the present invention is used for the preparation of a medicament for treating skin cancer, which is administered an effective dose of Ugonin compound to the skin to promote cell cycle arrest, regulate the expression and promotion of cell cycle related genes and proteins thereof. The expression of apoptosis-related genes and their proteins, and reduces the migration and invasion ability of the cancer cells; wherein the Ugonin compound is selected from Ugonin J or Ugonin L, and the Ugonin compound can be isolated, for example, from the rhizome of Ceylon be made of.

In one embodiment of the present invention, the cell cycle-related gene or protein is selected from the group consisting of p53, p21, Cyclin A, Cyclin B1, Cdk 2, and Cdc 2; preferably, the Ugonin compound increases p53 and The performance of the p21 gene or protein can also reduce the performance of the Cyclin A, Cyclin B1, Cdk 2 and Cdc 2 genes or proteins.

In one embodiment of the invention, the apoptosis-related gene or protein may be, for example, caspase-9 and caspase-3.

In one embodiment of the invention, the Ugonin compound further inhibits the performance of MMP-2, MMP-7 and MMP-9.

Accordingly, the Ugonin compound can further serve as a pharmaceutical composition for treating skin cancer or slowing the development of skin cancer, thereby effectively inhibiting the growth, migration and invasion of skin cancer cells.

First Figure-A: Cell viability test of Ugonin compounds

First Figure-B: IC50 and IC80 Concentrations of Ugonin Compounds in Poison Cells

Figure II-A: Schematic diagram of apoptosis analysis of Ugonin compounds in BCC cells

Figure II-B: Quantification of apoptosis analysis of Ugonin compounds in BCC cells

Fig. 3A: Schematic diagram of apoptosis analysis of Ugonin compounds in SCC25 cells

Figure III-B: Quantification of apoptosis analysis of Ugonin compounds in SCC25 cells

Figure 4A: Schematic diagram of cell cycle analysis of Ugonin compounds in BCC cells

Figure 4 - B: Quantification of cell cycle analysis of Ugonin compounds in BCC cells

Figure 5 - A: Schematic diagram of cell cycle analysis of Ugonin compounds in SCC25 cells

Figure 5 - B: Quantification of cell cycle analysis of Ugonin compounds in SCC25 cells

Figure 6 - A: Schematic diagram of Ugonin compounds in regulating BCC cell cycle-related gene expression

Figure 6 - B: Schematic representation of Ugonin compounds in regulating BCC cell cycle-associated protein expression

Figure 7 - A: Schematic diagram of Ugonin compounds in regulating the expression of SCC25 cell cycle-related genes

Figure 7 - B: Schematic diagram of Ugonin compounds in regulating the expression of SCC25 cell cycle-associated proteins

Figure 8 - A: Schematic diagram of Ugonin compounds in regulating the expression of apoptosis-related genes in BCC cells

Figure 8 - B: Schematic diagram of Ugonin compounds in regulating the expression of apoptosis-related proteins in BCC cells

Figure IX-A: Schematic diagram of Ugonin compounds in regulating the expression of apoptosis-related genes in SCC25 cells

Figure 9 - B: Schematic diagram of Ugonin compounds in regulating the expression of apoptosis-related proteins in SCC25 cells

Figure 11 - A: Schematic diagram of the migration ability of Ugonin compounds in cancer cells

Figure 11 - B: Quantification of Ugonin compounds in cancer cell migration analysis

Figure 11 - A: Schematic diagram of Ugonin compound in cancer cell invasion

Figure 11 - B: Schematic diagram of the inhibition ability of Ugonin compounds in matrix metalloproteinases

The object of the present invention and its structural and functional advantages will be explained in conjunction with the specific embodiments according to the structure shown in the following drawings, so that the reviewing committee can have a more in-depth and specific understanding of the present invention.

The invention relates to a Ugonin compound for preparing a medicament for treating skin cancer, which comprises administering an effective dose of Ugonin compound to the skin to promote cell cycle arrest, regulate cell cycle related genes or protein expression, and promote apoptosis related genes or proteins. The performance and the ability to reduce the migration and invasion of cancer cells for the treatment of skin cancer; wherein the Ugonin compound can be isolated, for example, from the rhizome of Helminthostachys zeylanica , and the Ugonin compound is selected from Ugonin. J or Ugonin L.

In one aspect of the invention, the Ugonin compound increases the expression of the p53 and p21 genes or proteins and decreases the expression of Cyclin A, Cyclin B1, Cdk 2 and Cdc 2 genes or proteins; the apoptosis-related gene or protein is caspase -9 and caspase-3; Ugonin compounds can further inhibit the performance of MMP-2, MMP-7 and MMP-9; accordingly, Ugonin compounds can be further used as a pharmaceutical composition for treating skin cancer or slowing the development of skin cancer, effectively It inhibits the growth, migration and invasion of skin cancer cells.

In addition, the scope of the invention may be further exemplified by the following specific examples, which are not intended to limit the scope of the invention.

<Ugonin Compound Source>

The dried rhizome of C. sylvestris ( H. zeylanica ) is pulverized, soaked in methanol, and then separated and purified by partition extraction, column chromatography and high performance liquid chromatography to obtain a chemical formula (I). A compound of Ugonin J (molecular weight 422), Ugonin K of chemical formula (II) (molecular weight 436) and Ugonin L of chemical formula (III) (molecular weight 436). The extraction steps and experimental parameters of Ceylon seven-finger fern can be implemented according to the paper "Research on Anti-inflammatory Active Components of Rhizome of Ceylon Seven Finger Fern" under the guidance of Prof. Chen Shengzhi of China Medical University, which is the technical field of the present invention. Those who have the usual knowledge can understand, so I won't go into details here.

Experiment 1: Cytotoxicity analysis of Ugonin compounds in human cancer cell lines and normal cells

Cell viability test

(1) Survival rate test of Ugonin compound in normal cells

Human breast epidermal cells (HBL100), human skin keratinocyte strain (HaCaT) or human fibroblast strain (Hs68) with a cell number of 1×10 ⁵ /mL were cultured in a 96-well plate at 37 ° C and 5% CO _{2 .} The cells were cultured for 24 hours in an incubator, and Ugonin J, K, and L at different concentrations (0 to 25 μM) were added and reacted in a 37 ° C, 5% CO ₂ incubator for 72 hours. After the reaction time, the culture solution was removed, and after washing with PBS, the new culture solution was replaced, and 10 μL of MTT (3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) solution was added. four hours later, the wavelength of measured absorbance ^{(BioTek, Synergy TM 2, USA} ) at 490nm.

(2) Survival rate test of Ugonin compound in cancer cells

Human lung adenocarcinoma cell line (A549), human prostate cancer cell line (DU-145), human liver cancer cell line (Hep3B), human skin basal cell carcinoma cell line (BCC) with a cell number of 1×10 ⁵ /mL The human breast cancer cell line (SKBR3) was cultured in a 96-well plate and cultured in a 37 ° C and 5% CO ₂ incubator for 24 hours. Ugonin J, K, and L at different concentrations (0 to 25 μM) were added at 37 ° C, 5 The reaction was carried out in a % CO ₂ incubator for 72 hours. After the reaction time was reached, the culture solution was removed, and after washing with PBS, a new culture solution was replaced, and after adding 10 μL of the MTT solution, the absorbance was measured at a wavelength of 490 nm. The dose concentration (μM) of 50% (IC ₅₀ ) inhibition of cell growth for different concentrations of the drug was calculated.

The results are shown in Tables 1 and 2. Ugonin compounds have obvious cytotoxic effects on BCC cell lines with IC ₅₀ values of 5.4, 7.5 and 11.1 μM, respectively. It is speculated that the Ugonin compound is selective for the toxicity of human skin cells, and the concentration of IC ₅₀ is higher in human normal skin cells than in skin cancer cells, indicating that Ugonin compounds are less toxic to normal skin cells.

It is known from the above results that Ugonin J, K, and L do not cause toxicity to normal skin cells in a short period of time, but do produce a high cytotoxic effect on skin cancer cells at the same dose. Therefore, in order to understand the toxic effects of Ugonin J, K, L on different types of skin cancer cell lines, human skin cancer cell lines including human non-melanoma skin cancer cell lines (BCC, SCC9, SCC25 and A431), human melanoma Cell line (A375) and control cell human skin keratinocyte cell line (HaCaT) and human skin fibroblast cell line (Hs68) were used to inhibit the growth of skin cancer cells by Ugonin J, K, L, and calculate the cells. The strain kills cells at 50% (IC ₅₀ ) and ₈₀ % (IC ₈₀ ).

The results are shown in Figure I-A and Figure I-B. Ugonin J, K and L have a toxic effect on human skin cancer cell lines, among which BCC and SCC25 cell lines are most significant, and cell viability (cell viabbility) It is the lowest and is less toxic to HaCaT and Hs68 cell lines. Therefore, the tumor cell cytotoxicity mechanism will be further explored for Ucanin J, K, and L for skin cancer cells BCC and SCC25.

Experiment 2: Apoptosis analysis of Ugonin compounds in BCC and SCC25 cells

1. <Annexin V-FITC / PI double staining analysis>

To confirm that Ugonin compounds may induce cancer cell death by apoptosis, apoptosis analysis was performed by flow cytometry using the Annexin V-FITC/PI double staining assay.

A cell volume of 1 x 10 ⁵ /mL was cultured in a 24well dish for at least 24 hours, after which Ugonin compounds of IC ₅₀ and IC ₈₀ concentrations were separately added and reacted in an incubator for 48 and 72 hours. The supernatants were separately collected into a 15 mL centrifuge tube, and the cells were removed into a centrifuge tube with trypsin-EDTA solution, centrifuged at 1,200 rpm for 5 minutes, and the supernatant was removed and 800 μL of binding buffer (containing 10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl ₂ ), the cells were evenly mixed, and 5 μL of Annexin V was added for 10 minutes. After adding 10 μL of propidium iodide (PI), the reaction was protected from light for 5 min, 800 μL of binding buffer was added, and evenly mixed. It was analyzed by flow cytometry; the fluorescence of Annexin V-FITC/PI double staining was in a four-way diagram, and the I region was a normal viable cell; the second region was a cell DNA fragmentation necrotic characteristic cell; the third region was a cell DNA fragmentation necrotic characteristic cell; It indicates that the cells are characterized by DNA fragmentation and late apoptotic necrotic cells; in the IV region, early apoptotic cells are distributed.

The results are shown in Figure II-A and Figure II-B. When Ugonin compounds act on BCC cells, the initial cells are distributed in the early apoptotic region of the IV region, but with the increase of the concentration of the effect and the prolongation of the reaction time, the cell distribution gradually enters the late phase. Dead necrosis stage (III zone); please refer to the third figure-A and the third figure-B. When Ugonin compound acts on SCC25 cells, SCC25 cells are initially distributed in the DNA fragmentation necrosis area of zone II, but increase with the reaction time. Late apoptotic necrosis (region III). It is concluded that Ugonin compounds promote BCC cell death in a cell-like manner, while SCC25 cells may be regulated by cell necrosis or autophagy in the early stage, but the cell apoptosis process is initiated at a later stage to cause cell death.

Experiment 3: Cell cycle regulation of Ugonin compounds in BCC and SCC25

<Cell Cycle Analysis>

To further explore the cell cycle regulation by Ugonin compound and related molecules cause cell toxic effect, the IC ₅₀ concentrations of compound Ugonin effect after BCC and SCC25 cells 24, 48 and 72 hours, the cells were transfected using DNA PI, and not to Drug-treated cells were compared as control groups and analyzed by flow cytometry.

A cell volume of 1.5 x 10 ⁵ /mL was cultured in a 24well dish for at least 24 hours, after which an IC ₅₀ concentration of Ugonin compound was added and reacted in an incubator for 24, 48 and 72 hours. The supernatant was separately collected into a 15 mL centrifuge tube, and the cells were removed into a centrifuge tube with trypsin-EDTA solution, and centrifuged at 1,200 rpm for 5 minutes with the supernatant. The supernatant was removed, 300 μL of PBS was added, the mixture was shaken slowly, and 700 μL of absolute alcohol was added dropwise to fix the cells, and then transferred to a microcentrifuge tube and stored in a refrigerator at 4 °C. Before flow cytometry, the cells were centrifuged at 1,200 rpm for 5 minutes at 4 ° C. The supernatant was removed and 445 μL PBS, 5 μL RNase (10 mg/mL), 50 μL, 10% Triton X-100 were added. After disrupting the RNA destruction of the cells, the reaction was carried out at 37 ° C for 30 minutes, centrifuged at 1,200 rpm for 5 minutes, the supernatant was removed, 400 μL of PBS was added and mixed uniformly, and 5 μL of PI (5 mg/mL) was added thereto, and the reaction was avoided at 47 ° C in the dark. In minutes, filter with a filter membrane. The flow cytometer (FACScan) was used to analyze the distribution of cell cycle with Winmdi computer software, and the cell cycle distribution after administration of the drug was observed.

The results are shown in Figure 4 -A and Figure 5 -A. After the Ugonin compound was applied to the BCC and SCC25 cell lines, the G ₀ /G phase ₁ ratio was decreased, and the S and G ₂ /M phases were controlled with the control group. In comparison, the ratio is significantly increased and the sub-G ₁ ratio is also increased. Therefore, it is inferred that the Ugonin compound acts to influence the SG ₂ /M phase and is sensitive to cells in the SG ₂ /M phase, thus arresting this period to induce apoptosis. The cell cycle ratio was quantitatively analyzed, and the results are shown in Fig. 4B and Fig. 5B. The sub-G ₁ of BCC and SCC25 cells without drug treatment was only 0.5% and 1.5%. After treatment with Ugonin J, K, L for 72 hours, the percentage of sub-G ₁ BCC cells was 40.1%, 47.3%, 35.4%. SCC25 cells were 27.9%, 27.9%, and 43.9%. From the above results, Ugonin compounds affect the cell cycle of BCC and SCC25, and induce apoptosis of cancer cells.

Experiment 4: Cell cycle related genes and protein expression of Ugonin compounds in BCC and SCC25

It can be seen from the cell cycle test that when BCC and SCC25 cells are subjected to Ugonin compounds, the cells are arrested in the SG ₂ /M phase to further cause apoptosis. Therefore, the expression of regulatory factors related to the cell cycle is further explored.

1. Polymerase chain reaction (PCR) analysis of gene expression

(1) Extraction of cellular RNA

The cell volume of 1.5 × 10 ⁵ /mL was cultured in a 24-well plate for at least 24 hours, after which an IC ₅₀ concentration of Ugonin compound was separately added and reacted in an incubator for 24, 48 and 72 hours. After collecting the cells and washing twice with PBS, the supernatant was removed. Add 1mL REzol ^TM C & T solution, 25 ℃ for 5 minutes to dissolve the cells. Then, 200 μL of chloroform was added, mixed well and placed on ice for 5 minutes. After centrifugation at 12,000 rpm for 15 minutes at 4 ° C, the supernatant was taken to another tube of a new microcentrifuge tube, and an equal volume of ice isopropanol was added and mixed for 10 minutes. After centrifugation at 12,000 rpm for 15 minutes at 4 ° C, the RNA will form a clear white pellet at the bottom of the microcentrifuge tube. The supernatant was removed, and the precipitate was gently rinsed with 200 μL of ice 75% alcohol, and after centrifugation at 12,000 rpm for 5 minutes at 4 ° C, the supernatant was removed. The air-dry RNA pellet was allowed to stand for 15 minutes, after which the RNA pellet was dissolved in an appropriate amount of DEPC-treated H ₂ O, stored at -80 ° C, and then the absorbance was measured spectrophotometrically at OD ₂₆₀ nm and OD ₂₈₀ nm. .

(2) Preparation of cDNA by reverse transcription (RT)

Take 2 μg of RNA, add appropriate amount of diethyl pyrocarbonate (DEPC) water, add 1 μL of Oligo (dT) ₁₈ primer at 70 ° C for 2 minutes, then quickly transfer to ice, then add 4 μL of 10 × MMLV RT buffer solution, 1 μL dNTP mixture ( 10 mM each dNTP), 0.5 μl of recombinant RNase inhibitor (1 unit/mL), and 1 μl of MMLV reverse transcriptase (5 unit) to a total volume of 20 μL. After mixing uniformly, the mixture was allowed to act at 42 ° C for 1 hour, and then heated at 94 ° C for 5 minutes to remove the MMLV-RTase activity and terminate the reaction, that is, the preparation of the first strand of cDNA was completed, and 80 μL of DEPC water was added and stored at -20 ° C.

(3) Polymerase chain reaction (PCR)

Take 1 μL of cDNA into a microcentrifuge tube and add 5 μL of 10×reaction buffer, 0.8 μL of 10 mM dNTPs (200 mM each dGTP, dATP, dTTP and dCTP) and 1 μL of 50 mM upstream primer, downstream primer, Taq DNA polymerase (5 unit/μL). Finally, deionized water (ddH ₂ O) was added to bring the total volume to 50 μL. After mixing uniformly, the PCR reaction was carried out in an automatic temperature cycle machine (Techne Progene) under the following conditions: a denaturation of 98 ° C, a cycle of 3 minutes; followed by a denaturation reaction of 94 ° C, an annealing (60 ° C) The synthesis reaction (extension) was carried out at 72 ° C for 1 minute for the polymerase chain reaction, and the total reaction process was carried out for 35 cycles. After completion of the reaction, an appropriate amount of the reaction product was analyzed by 2% agarose gel electrophoresis while performing 100 volt electrophoresis. After the DNA was run to an appropriate distance, the gel was taken out, observed on an ultraviolet transilluminator, and photographed for storage. Among them, the introduction of cell cycle related genes is shown in Table 3.

<Western blotting analysis of protein expression>

3×10 ⁵ /mL cells were cultured in 10 cm culture dishes and cultured in 37 ° C and 5% CO ₂ incubator for at least 24 hours. After adding 10 μM Ugonin compound for 4 hours, UVB (50 mJ/cm ² ) was irradiated, and the serum-free medium was changed for the reaction time. After the cells were collected by Trypsin-EDTA, the cells were collected by centrifugation at 1,200 rpm for 5 minutes to remove the supernatant. Then, the cells were transferred to a microcentrifuge tube, 100 μl of RIPA solution was added to completely dissolve the cells, and then centrifuged at 12,000 rpm for 10 minutes, the supernatant was collected, and BSA protein was used for protein quantitative analysis, and the protein extract was stored at -80 ° C. . 40 μg of the protein sample was taken for SDS-PAGE electrophoresis, and then the protein on the gel was transferred to a polyvinylidene difluoride (PVDF paper) by a transfer tank. In a transfusion buffer [25 mM Tris, 192 mM glycine, 20% (v/v) methanol, pH 8.3], a constant voltage of 200 was applied for 2 hours. The PVDF paper was covered with 5% skim milk powder in PBS/Tween-20 (PBST) buffer for the purpose of blocking the non-specific binding sites on the PVDF paper and reacting at room temperature for 30 to 60 minutes. The blocking buffer was then removed and the diluted primary antibody was added and reacted at 4 ° C until overnight. After the primary antibody reaction was combined, the PVDF paper was washed three times for 5 minutes in PBST buffer. The diluted secondary antibody was then added, shaken slowly for 2 hours at room temperature, and the PVDF paper was washed five times with PBST buffer for five minutes each. After the final washed PVDF paper was added to the ECL luminescence-based detection system, the luminescence system was used to photograph and analyze the expression of the protein to be measured in the cells. Among them, the antibodies of cell cycle related proteins are shown in Table 4.

The results are shown in Figure 6-A. After 24, 48 and 72 hours of treatment with Ugonin compounds in BCC cell lines, the expression of related genes such as p53 and p21 was significantly increased before the cell cycle, and the cell cycle was in SG. _{During the 2} /M period, a large number of stagnant accumulations, which in turn reduced Cyclin A, Cdk 2, Cyclin B1 and Cdc 2 performance. Western blotting analysis of BCC cell cycle-related protein performance results, as shown in Figure 6-B, protein p53 and p21 performance increased, while Cyclin A, Cdk 2, Cyclin B1 and Cdc 2 showed a downward trend. It was confirmed that the action of Ugonin compounds affects the expression of cell cycle regulatory proteins, which in turn causes the cell cycle to stagnate, leading to growth arrest.

Please refer to Figure 7A again. After 24, 48 and 72 hours of treatment with Ugonin compounds, the expression of p53 and p21 was significantly increased before the cell cycle. The cell cycle was in the SG ₂ /M phase. A large amount of stagnant accumulation, which in turn reduces Cyclin B1 and Cdc 2 performance. Western blotting analysis of SCC25 cell cycle-associated protein performance results As shown in Figure 7-B, protein p53 and p21 showed an increasing trend while Cyclin A, Cdk 2, Cyclin B1 and Cdc 2 showed a downward trend. It was confirmed that the action of Ugonin compounds affects the expression of cell cycle regulatory proteins, which in turn causes the cell cycle to stagnate, leading to growth arrest.

Experiment 5: Effect of Ugonin Compounds on the Regulation of Caspases Apoptosis Protein in BCC and SCC25 Cell Lines

Previous experiments by Annexin V-PITC/PI double staining analysis confirmed that the cell death caused by Ugonin compounds may regulate the death of both autophagy and apoptosis, so the apoptosis regulation was further analyzed by RT-PCR and Western blotting. The genes and proteins of the factors caspase-8, caspase-9, caspase-3, bid, and PARP are expressed; among them, the primers of the apoptosis-related genes and the antibodies of related proteins are shown in Table 5 and Table 6, respectively.

The concentration of ₅₀ to act on the compound IC Ugonin BCC cells, performed RT-PCR. 0,24, 48 and 72 hours apoptotic caspases gene expression assay, the results shown in the eighth FIG -A, and the control group (0h) The expression of caspase-8, caspase-9, caspase-3 and bid genes was increased compared with Ugonin compounds. Furthermore, Ugonin compounds at IC ₅₀ concentration were applied to BCC cells for 48 and 72 hours, followed by Western blotting. The expression of related proteins was measured. The results were as shown in Figure 8-B. Compared with the control group (0h), Caspase-8, Caspase-9 and Caspase-3 were significantly increased and Bid was activated by Ugonin compounds. The t-Bid and the PARP protein downstream of the apoptotic pathway are clearly cleaved (cleaved PARP), which loses the ability to repair DNA and causes DNA cleavage and fragmentation to induce apoptosis. Therefore, it was confirmed that Ugonin compounds can regulate the expression of activation bid and promote the activation of caspase, and then PARP is activated by shear, leading to apoptosis of BCC cells.

The concentration of ₅₀ to act on the compound IC Ugonin SCC25 cells, RT-PCR to be 24, 48 and 72 hours apoptotic caspases gene expression measurement results are shown in FIG -A ninth, control group (0h) compared The expression of caspase-9 and caspase-3 was increased by Ugonin compound, but there was no significant change in caspase-8 and bid gene expression. Furthermore, Ugonin compound at IC ₅₀ concentration was applied to SCC25 cells for 48 and 72 hours. Western blotting method was used to determine the related protein expression. The results were as shown in Figure IX-B. Compared with the control group (0h), the expression of Caspase-8, Caspase-9 and Caspase-3 was observed by Ugonin compound. The 48-hour increase, the activation state of t-Bid increased, and the PARP protein downstream of the apoptotic pathway was cleaved (cleaved PARP). However, its protein expression is not obvious compared to BCC. Therefore, it is concluded that Ugonin compounds can regulate the expression of activated caspase for SCC25 but not completely cause apoptosis in cancer cells.

Experiment 6: Inhibition of cell migration by Ugonin compounds in A375 and SCC25 cells

The main feature of cancer cells is that they can invade the surrounding tissues, and can migrate to other tissues in the distance, and then grow uncontrollably, eventually leading to death. Therefore, in addition to killing cancer cells, the inhibition of the spread of migratory cancer cells is also an important direction in the development of anticancer drugs. In this experiment, the most migratory A375 and SCC25 cell lines in skin cancer were selected for cell migration assay. The cells were treated with Ugonin at different time points for 0, 24 and 48 hours, and the cell migration was analyzed by wound healing experiments.

<Cell cancer migration ability test>

Culture-insert was placed in a 24-well disc culture dish, and a cell volume of 3 x 10 ⁴ /70 μL was injected into the cells in both chambers for at least 24 hours to allow cell spacing. Thereafter, the Ugonin compound at an IC ₂₀ concentration was added, and after reacting for 24 and 48 hours in an incubator, photographing was performed by an optical microscope, and the cell spacing was calculated.

<Invasion ability test>

The 8 μm insert filter was mounted on a 24-well plate, then 30 μg of basement membrane matrix was added to the filter, placed at 37 ° C for 1 hour, and then the cells (5 × 10 ⁴ /100 μL) were seeded onto the membrane, IC ₂₀ The concentration of Ugonin compound was incubated at 37 ° C for 48 hours. The upper layer of cells was scraped off with a cotton swab to retain the cells at the bottom of the filter membrane. The cells were fixed with 4% formaldehyde. After staining with 0.1% crystal violet, the filter was counted. The cells at the bottom.

For the results, please refer to the tenth figure-A and the tenth figure-B. The control cell spacing (Gap distance) of A375 and SCC25 is 130.0 and 126.6μm. After 48 hours, the cell spacing is shortened to 44.6 and 88.6μm. It showed that the migration ability of A375 cells was better than that of SCC25, and the cells treated by the compounds Ugonin J, K, L had obvious effects of inhibiting cell migration; the cell spacing A375 for 48 hours was 127.3, 122.0 and 108.0 μm, respectively; SCC25 was 124.6, 120.0 and 113.3 μm. In particular, A375 cells were significantly inhibited by the Ugonin compound under the 48 hour treatment compared to the control group. Ugonin compounds were shown to inhibit the migration of A375 and SCC25 cells.

Experiment 7: Inhibition of cell invasion assay and matrix metalloproteinases (MMPs) by Ugonin compounds in A375 cells

The invasion of cancer cells into the surrounding interstitial matrix is the key to the conversion of benign tumors into malignant tumors. Malignant tumor cells themselves produce extracellular matrix decomposing enzymes (such as matrix metalloproteinases, MMPs) that break down the basement membrane and are made up of blood vessels. Infiltrate the tissue to achieve the purpose of migration. Among them, MMP-2, MMP-7 and MMP-9 Over-expression of the basement membrane is most correlated with cancer invasion and migration, so the detection of MMPs in cancer cells is used as an indicator of local invasion and distant migration.

The difference in the degree of invasion of Ugonin compounds in A375 cells was analyzed by transwell chamber coating Matrigel. The results are shown in Fig. 11-A. Compared with the control group, A375 cells can effectively inhibit the invasion of A375 cells by Ugonin compounds, especially the number of cells acting on the lower layer of the transwell chamber of Ugonin K group, inhibiting A375. The ability to invade cells is optimal. The protein expression results of matrix metalloproteinases MMP-2, MMP-7, and MMP-9 are shown in Fig. 11-B. Compared with the control group, after 24 hours, MMP-2, MMP-7 and MMP The activity of -9 showed a decreasing trend. The results confirmed that Ugonin compounds can inhibit the invasion and migration of A375 cancer cells, and Ugonin compounds will help to develop drugs to inhibit tumor growth, invasion and migration.

In summary, Ugonin compounds specifically kill cancer cells, promote skin cancer cell apoptosis and slow the development of cancer cells (migration or invasion), so Ugonin compounds can be used alone or in combination with pharmaceutically acceptable carriers or excipients. It is applied as a pharmaceutical composition for preparing or slowing skin cancer; providing a user with an appropriate amount to apply to the skin, thereby achieving the effect of inhibiting migration and invasion of skin cancer cells. The "pharmaceutically acceptable carrier or excipient" as used in the present invention includes, but is not limited to, water, physiological saline, organic solvents, stabilizers, chelating agents, preservatives, emulsifiers, suspending agents, diluents, Gels, liposomes, and the like.

It can be seen from the above description of the implementation that the present invention is compared with the prior art. Ming has the following advantages:

1. The present inventors first confirmed the use of Ugonin compounds in the treatment of skin cancer by experiments, whereby Ugonin compounds can further serve as a pharmaceutical composition for the treatment of skin cancer cells, effectively killing cancer cells and promoting cell cycle arrest in SG ₂ / M phase, and stop cancer cell migration and invasion.

2. The present inventors have also experimentally confirmed that Ugonin compounds can induce apoptosis mechanism of skin cancer cells to promote cancer cell death. It is expected that Ugonin compounds have the potential to develop effective and specific anti-skin cancer drugs, and provide for skin cancer. Good treatment.

3. Traditionally used drugs for the treatment of cancer can also damage normal cells. Ugonin compounds are more toxic to skin cancer cells, but less toxic to normal skin cells, which can reduce the doubts that drugs affect normal cell function.

4. In the past, Chinese medicine practitioners have the effects of antipyretic, anti-inflammatory, analgesic, and myogenic effects on the efficacy of Ceylon seven-fin fern. However, the present invention proves that the Ugonin compound derived from the rhizome portion of the C. sylvestris can be applied to the anti-skin cancer effect, and finds a new effect which has not been found in the past.

In summary, the use of the Ugonin compound of the present invention for the preparation of a medicament for treating skin cancer can indeed achieve the intended efficacy by the above-disclosed examples, and the present invention has not been disclosed before the application, and has been completely completed. Meet the requirements and requirements of the Patent Law.爰Issuing an application for a patent for invention in accordance with the law, and asking for a review, and granting a patent, is truly sensible.

However, the illustrations and descriptions disclosed above are only preferred embodiments of the present invention, The scope of the present invention is defined by the scope of the invention, and other equivalents and modifications of the invention are intended to be within the scope of the invention.

Claims (6)

A Ugonin compound for the preparation of a medicament for treating skin cancer, which is administered an effective dose of Ugonin compound to the skin to promote cell cycle arrest, regulate cell cycle related genes or protein expression, and promote expression of apoptosis-related genes or proteins. And reduce the ability of cancer cells to migrate and invade, wherein the Ugonin compound is Ugonin J or Ugonin L. The use of the Ugonin compound according to claim 1, wherein the cell cycle-related gene or protein is selected from the group consisting of p53, p21, Cyclin A, Cyclin B1, Cdk 2 and Cdc 2 . Group. The use of a Ugonin compound according to claim 2 for the preparation of a medicament for treating skin cancer, wherein the Ugonin compound increases the expression of the p53 and p21 genes or proteins. The use of a Ugonin compound according to claim 2 for the preparation of a medicament for treating a skin cancer, wherein the Ugonin compound reduces the expression of the Cyclin A, Cyclin B1, Cdk 2 and Cdc 2 genes or proteins. The use of a Ugonin compound according to claim 1 for the preparation of a medicament for treating skin cancer, wherein the Ugonin compound further inhibits the expression of MMP-2, MMP-7 and MMP-9. The use of a Ugonin compound according to claim 1 for the preparation of a medicament for treating skin cancer, wherein the Ugonin compound is isolated from the rhizome portion of Helminthosachys zeylanica .