Cancer Research

RECNAC , Fatty Acid , Lipoic-Acid , Natural Extracts , Bindweed , LF Bacterium

Anticancer Effect of Natural Organism Extracts:
The natural organisms include plants, bacteria and invertebrate. The purpose of this study is to find a high effective but low toxic anticancer agent.

Here is how we did the study. The natural organisms are minced and soaked by buffers or solvents. After precipitation, centrifugation, filtration and lyophilization, we get a crude extract. The crude extract will be analyzed by spectrophotometry, chromatography, electrophoresis, etc.

It will be tested on cultured cells for cytotoxicity, on eggs for angiogenesis, on animal models for tumor inhibition. If the crude extract shows good results, we may separate it by the polarity, size and charge of the molecules and repeat the analysis and the above tests until we get the high effective and low toxic anticancer agent.

This slide shows the differential cytotoxicity of an invertebrate extract on normal and tumor cells. CCD is a colon normal cell line. LS is a colon cancer cell line. The others are tumor cell lines too. Y axis is the cell growth rate. The invertebrate extract kills tumor cells 15 times more than normal cells. This is one way that we look for the effective molecules in vitro: the high differential cytotoxicity.

This slide shows the dose responses of a plant extract on sarcoma and lung cancer cell lines. The upper left is the sarcoma cells. The lower right is the lung cancer cells. X axis is the dose, µg/ml. Y axis is the cell growth inhibition. This plant extract is toxic only at very high concentration, 1000 or 2000 µg/ml, while the chemotherapeutic agents are usually less than 10 µg/ml.

The tumor inhibition of this extract on animal test (70-80%) was definitely not due to the direct tumor toxicity. This fit our critearia of high effect and low toxicity.

This slide shows the angiogenesis inhibition in cultured cells. Angiogenesis means new blood vessel starting and growing. It happens around all malignent tumors. If any drug can inhibit the angiogenesis around a tumor, it may stop the growth and metastasis of the tumor.

Capillary blood vessels consist of endothelium cells. ECV304 is an endothelium cell line. An agent that inhibit ECV304 cells in vitro may inhibit angiogenesis in vivo. At 10 µg/ml level, one of our bacterium extract showed 60% inhibitio and a good dose response. In the literature, 30% inhibition was a good one for the angiogenesis inhibition at 10 µg/ml level.

Cell culture is a fast but indirect experiment for studying angiogenesis. Using the fertilized eggs, we can see the angiogenesis and its inhibition directly.

This slide shows the eggs we used for the angiogenesis study. Some angiogenesis stimulator, such as tumor cells or heparin, were applied to the eggs. The tested agents were applied to the eggs too to see the angiogenesis inhibitioin.

This slide shows the eggs we used for the angiogenesis study. Some angiogenesis stimulator, such as tumor cells or heparin, were applied to the eggs. The tested agents were applied to the eggs too to see the angiogenesis inhibitioin.

The left picture of this slide shows the angiogenesis. Many capillary blood vessels grow around a stimulator. The right picture shows the angiogenesis inhibition by one of our plant extract (BWR). Capillaries were much less around the stimulator. This is the other way to find the effective molecules for cancer treatment.

The left picture of this slide shows the angiogenesis. Many capillary blood vessels grow around a stimulator. The right picture shows the angiogenesis inhibition by one of our plant extract (BWR). Capillaries were much less around the stimulator. This is the other way to find the effective molecules for cancer treatment.

We also studied the tumor inhibition on the egg model. HT1080 fibrosarcoma cells can induce a solid tumor in eggs. The left picture of this slide shows the egg tumor induced by HT 1080 cells. The weight of the tumor is 90-100 mg. It caused bleeding if it kept growing for 7 days.

We also studied the tumor inhibition on the egg model. HT1080 fibro sarcoma cells can induce a solid tumor in eggs. The left picture of this slide shows the egg tumor induced by HT 1080 cells. The weight of the tumor is 90-100 mg. It caused bleeding if it kept growing for 7 days.

Under the microscope, the cells from the HT1080 induced tumor were big and the nucleus were big too. The left picture of this slide shows the cells from the non-tumor induced clump. The right picture shows the cells from the HT1080 induced clump. Big cells and nucleus mean active metablism. These are the features of tumor cells. All these indicate that a real tumor was induced by the HT1080 cells in the eggs.

Under the microscope, the cells from the HT1080 induced tumor were big and the nucleus were big too. The left picture of this slide shows the cells from the non-tumor induced clump. The right picture shows the cells from the HT1080 induced clump. Big cells and nucleus mean active metabolism. These are the features of tumor cells. All these indicate that a real tumor was induced by the HT1080 cells in the eggs.

The left picture of this slide shows the HT1080 cell induced tumor and angiogenesis around the tumor. The right picture shows the tumor and angiogenesis inhibition of one of our invertebrate extract. The tumor was much smaller. The blood vessels was less.

During and after the above experiments, we may separate and purify the crude extracts further. This slide shows one of our chromatography systems. Some of the crude extracts were analyzed and purified on this system.

During and after the above experiments, we may separate and purify the crude extracts further. This slide shows one of our chromatography systems. Some of the crude extracts were analyzed and purified on this system.

This slide shows a chromatogram of a plant extract from the above system. The peaks can be collected separately so that they can be purified.

This slide shows a chromatogram of a plant extract from the above system. The peaks can be collected separately so that they can be purified.

This slide shows the tumor inhibition of plant extracts on the mouse tumor models. The green color is for control group. The red color is for the treating group. Y axis is the tumor weight. In control groups, the tumors were very big.

This slide shows the tumor inhibition of plant extracts on the mouse tumor models. The green color is for control group. The red color is for the treating group. Y axis is the tumor weight. In control groups, the tumors were very big.

In treating groups, due to the anticancer agents, the tumors were much smaller. For the sarcoma model, the tumor inhibition is 77%. For the lung cancer, the inhibition is 62%. For the melanoma, the inhibition is 53%. On these animal models, tumors grow very fast. In two or three weeks, the tumor can grow 8-10% of the body weight. If a tested agent can reach 30-40% tumor inhibition on these models, it will be considered a hopeful anticancer agent. Some of our agents reached more than 80%.

These extracts showed not only very high tumor inhibition, but also low toxicity. The left chart of this slide shows the tumor inhibition of a bacterium extract on the sarcoma model. The tumor inhibition was 77%. The right chart of this slide shows the comparison of the toxic and treating dosages.

The right chart of this slide shows the comparison of the toxic and treating dosages. The LD50 means 50% lethal dosage. It was 4293 mg/kg for the bacterium extract. While the daily dosage for the 77% tumor inhibition was only 12.5 mg/kg. That is 0.3% of the LD50. Usually 10% or below of the LD50 is the safe dosage used in clinic. So the toxicity of this bacterium extract was very low. We did the LD50 for another plant extract. The toxicity was also very low.

The LD50 means 50% lethal dosage. It was 4293 mg/kg for the bacterium extract. While the daily dosage for the 77% tumor inhibition was only 12.5 mg/kg. That is 0.3% of the LD50. Usually 10% or below of the LD50 is the safe dosage used in clinic. So the toxicity of this bacterium extract was very low. We did the LD50 for another plant extract. The toxicity was also very low.