William Jia, PhD, Faculty of medicine, University of British Columbia

Reasons for losing battle against cancer

Cancer cells are genetically unstable. That means genes in cancer cells are constantly mutating while cells are proliferating. Consequently, cells even within the same tumor are different: this heterogeneity makes cancer extremely difficult to treat. Since current treatment is based on some biological characteristics of cancer cells, which may be only suitable for a cell subpopulation in a tumor. The remaining malignant cells are likely resistant to treatment due to their different biological characteristics. That is one of the major reasons why current therapy is generally ineffective. Another important reason for failure in cancer treatment is multidrug resistance (MDR) developed by cancer cells either inherited or induced by chemotherapy. Again, mechanisms for MDR vary from cell to cell, adding another layer of difficulty for treatment.

Criteria for an "IDEAL" anti-cancer drug

Based on the above considerations, an ideal anti-cancer drug should:

1) Attack all types of cancer cells
2) Non-toxic and allow long-term treatment
3) Not induce drug resistance

While an ideal anti-cancer drug does not exist yet, scientists have recently discovered a compound with several pharmacological properties that render it a potential ideal anti-cancer agent. This compound is named Dammarane sapogenins.

Development of dammarane sapogenines

Dammarane sapogenins are a group of compounds found in plants, especially in araliaceae plants. The dammarane sapogenins backbone is a tetracyclic terpene of the dammarane series (Fig. 1). While plants (such as ginseng) containing those compounds have been extensively used for medicinal use in China and other Asian countries for thousands of years, extracts do not shown significant cancer killing activity. Starting from the 1980’s, a protopanaxadiol compound Rh2, was shown to possess strong anti-cancer activity and Rh2 has been the focus of attention over the past two decades. However, the amount of Rh2 in plants is extremely low. In order to obtain one gram of Rh2 hundreds of raw ginseng plants which has the highest Rh2 content among all plants would be needed. The extremely high cost of producing of Rh2 prevents it from becoming an anti- cancer drug candidate. Dammarane sapogenins are series of compounds derived from natural plants including ginseng, and produced through an advanced chemical technology. The chemical structures of dammarane sapogenins are similar to that of Rh2 and possess equal or more potent Rh2 analogs with anti-cancer activities. However, the cost is much lower than that of Rh2 rendering it a potential ideal anti-cancer candidate.

Major anti-cancer characteristics of dammarane sapogenins

Dammarane sapogenins act on cancer cells through the following pathways through a multi-targeting and multi-dimensional approach.

1. Dammarane sapogenins arrest cancer proliferation and induce cancer cell apoptosis through multiple mechanisms
2. Dammarane sapogenins as potential chemosensitizers
3. Dammarane sapogenins are potential cancer prevention agents

In animal models, dammarane sapogenins are able to inhibit cancer cell proliferation. In addition, dammarane sapogenins can kill a very wide range of cancer cells of different origins with varying genetic backgrounds, and can induce apoptosis of cancer cells using a multi-targeting and multidimensional approach.

1.1 Dammarane sapogenines inhibit tumor growth in animal models

To demonstrate the therapeutic efficacy of dammarane sapogenines, one must test it on animal tumor models before use on patients. An example is shown in fig. 1. Fig. 1 shows result of an experiment that used animals bearing malignant brain tumors. In this study, malignant glioma cells were implanted in the brain. Control animals only received saline while the experimental groups received dammarane sapogenins containing agent orally for 10 days. The results are shown with keplain-Meior surviving curves. The x-axis represents survival days and the y-axis is the percentage of animals that survived on each day. These survival curves show that animals in the control group all died within 24 days while 40% animals treated with dammarane sapogenins containing agent in the dose group 25mg/kg survived. In addition, even at a low dosage, the animals lived longer compared to the control group, demonstrated by a right shift of the survival curve.

As shown in fig. 2, tumor sizes of dammarane sapogenins treated animals were much smaller than in the control group suggesting an inhibition of tumor growth. A similar tumor inhibitory effect was also seen in prostate cancer and pancreatic cancer animal models.

Dammarane sapogenins can kill a very wide range of cancer cells of different origins with varying genetic backgrounds

Dammarane sapogenins and derivative compounds are effective against a very wide spectrum of cancer cell types. Every cancer cell tested can be killed by dammarane sapogenins regardless of tissue origin.

As shown in fig. 3, MCF-7 is a human breast cancer cell line, PC3 is human prostate cancer, SF188 is human brain cancer and 9L is rat brain tumor. All of these cells are responsive to dammarane sapogenins cytotoxicity within a narrow dose range. In addition, dammarane sapogenins also induce apoptosis in cancer cells of different genetic background.

Fig. 4 depicts an example where two types of brain tumor cells were treated with dammarane sapogenins. These two types of tumor cells had different genetic backgrounds, in particular, SF188 and U87 brain cancer cells lack two important genes termed p53 and PTEN, respectively. The p53 gene is a gene expression protein; normal expression of p53 plays an important role in cell cycle control and apoptosis. Mutation in p53 not only leads to cell transformation, but also confers resistance to chemotherapy since many anti-cancer drugs rely on normal functions of p53 for their therapeutic effects.

Mutation in the PTEN gene is also commonly seen in cancer cells. PTEN can regulate the survival pathway within cells by decreasing Akt activation. Scientists have found that lacking the PTEN gene may lead to the persistent patency of the intracellular survival pathway, conferring resistance against a variety of therapies. It is therefore important to note that dammarane sapogenins and its analogs can destroy cancer cells independent of their p53 and PTEN genetic status.

1.3 Dammarane sapogenins induce cancer cell apoptosis through multiple mechanisms
Apoptosis is a special form of cell death, also named programmed cell death. Fig. 5 shows a typical microphotograph of apoptotic cells, where clusters of small vesicles (apoptotic bodies) are seen in treated cancer cells. Dammarane sapogenins and derivatives may activate a series of enzymes inside cells in an orderly fashion and resulting in cancer death through activating apoptosis pathways.

As shown in fig. 6, dammarane sapogenins react with cancer cells and initiate the following mechanisms simultaneously to cause cancer apoptosis.

• activates multiple caspases and induces cancer cell apoptosis
• Dammarane sapogenins raise high levels of free radicals inside cancer cells to cause apoptosis
• inhibit the process of Akt phosphorylation, and shut down the survival pathway of cancer cells

Dammarane sapogenins activate multiple caspases and induce cancer cell apoptosis

Fig. 7 is a diagram of a signal transfer pathway in cells that carries a death signal initiating apoptosis. In this particular pathway, a group of enzymes known as caspases play crucial roles.

Caspases represent is a group of enzymes, which can initiate and execute apoptosis. Caspases induce abnormal cells to apoptosis through a cascade of internal signalling. As shown in fig.7, caspase 8 is an upstream player that transfers the initial death signal to caspase 3, or 6 or 7. Cytochrome C and caspase 9 play similar roles as caspase 8 as initiators to transfer the death message. Mutations in cancer cells often result in interruption of those signal transduction pathways to allow cancer cells to survive in the presence of death signals initiated with various anti-cancer drugs or therapies. Interestingly, sufficient evidence has demonstrated that dammarane sapogenins activate all of the caspases tested in many cancer cells in an almost simultaneous fashion including caspase 8, 9 and caspase 3, 6 and 7, the so-called death executors. Thus, for the mechanism involving caspases, dammarane sapogenins do not require any particular upstream caspase (caspase 8, 9) and are capable of “short-cutting” the death signal pathway to directly activate caspase 3, 6 or 7 to cause cancer cell death.

Dammarane sapogenins can also induce cancer apoptosis WITHOUT caspases

In some cancer cells, the caspase mechanism is not operative. To induce apoptosis, other mechanisms must be utilized.

• Dammarane sapogenins raise high levels of free radicals inside cancer cells causing apoptosis.

Current studies demonstrated that dammarane sapogenins are able to induce cancer cell apoptosis by generation of high levels of free radicals. It is well-known that free radicals may stimulate multiple genes and turn on a number of apoptosis pathways including caspase-dependent and caspase-independent mechanisms.

In our experiment, after treating cancer cells with dammarane sapogenins for 1 hour, most of the cancer cells generated high levels of free radicals.

• Dammarane sapogenins inhibit Akt phosphorylation and hinder the cancer cell survival pathway

Cancer cells are constantly under tremendous stress such as from attack by the patient's own immune system, and various anti-cancer drugs and therapies. It has been found that cancer cells are able to utilize a survival mechanism existing in every cancer cell to defend themselves from those insults. In normal cells, the survival pathway is usually turned off and is only transiently enabled when the cell is under stress. However, cancer cells are capable of maintaining the survival pathway active by disabling some controlling element such as the PTEN gene.

Akt is an anti-apoptotic enzyme that keeps this survival pathway patent and resists all kinds of anti-cancer therapies. It is therefore proposed that one may kill cancer cells by simply turning off the survival pathway. One way to turn off the pathway is through regulating the activity of Akt. Akt is activated through the process of phosphorylation. Activated Akt may enhance the proliferation of cancer cells, and also can inhibit apoptosis. Therefore, Akt plays a pivotal role in cancer cell survival.

Normal PTEN decreases Akt activity resulting in increased levels of apoptosis. In contrast, mutation of PTEN is associated with increased levels of Akt, and may hinder the apoptosis of cancer cells.

Dr. William Jia’s laboratory at the University of British Columbia recently discovered that dammarane sapogenins and analogs can effectively inhibit the survival pathway in PTEN negative cancer cells, in which this pathway is constitutively active. The results showed that when tumor cells were treated with dammarane sapogenins, the levels of phosphorylated Akt were substantially reduced. Furthermore, dammarane sapogenins can even shut off the production of Akt in some cancer cells, therefore completely disabling the survival mechanism in those cancer cells.

2. Dammarane sapogenins as a potential chemosensitizer
Cancer cells are often resistant to chemotherapy agents. This can be inherited due to genetic background or can be acquired during the course of chemotherapy. The mechanisms for cancer drug resistance are complex including abnormalities in apoptosis pathways and a constitutively active survival pathway. Dammarane sapogenins can work with other anti-cancer agents to enhance the efficacy of treatment and overcome the multidrug resistance of cancer cells to chemotherapy through the following mechanisms:

2.1 Dammarane sapogenins can inhibit P-gp protein

P-gp protein is located in the cancer cell membrane and functions as a pump that continuously pumps out any anti-cancer drugs from the inside of cancer cells. Thus, P-gp lowers the amount of drug inside of the preventing killing. Among all of the other drug resistant mechanisms, over-production of P-gp protein by cancer cells is one of the most important.

Studies in Dr. William Jia’s lab have demonstrated that dammarane sapogenins can effectively inhibit the function of P-gp to allowing much more drugs accumulation inside cancer cells. This is illustrated in fig. 9. The two panels are microphotographs of multidrug resistant human breast cancer cells. To test the activity of P-gp, the cells were incubated with a special dye that is florescent outside but becomes florescent once accumulated inside of cells. Fig. 9a shows cells treated with the dye only and fig. 9b are cells treated with dammarane sapogenins for 15 min before adding the dye. Clearly, dammarane sapogenins treated cells had high levels of dye accumulated inside of cells making every cell light up compared to the untreated cells. These results indicate that dammarane sapogenins can effectively disable the function of P-gp, which allows higher levels of chemotherapy drugs to accumulate inside of cancer cells to enhance the therapeutic efficacy.

2.2 Dammarane sapogenins work synergistically with chemotherapeutic agents and enhance its efficacy

Dammarane sapogenins not only can act alone to induce apoptosis but can also work synergistically to enhance the efficacy of chemotherapeutic agents. Furthermore, dammarane sapogenins can induce apoptosis and block the survival pathway to enhance cytotoxicity of chemotherapeutic agents to cancer cells.

• Dammarane sapogenins versus Taxol

In this experiment, human breast cancer cells with high levels of P-gp were divided into three groups. One group was treated with a common chemotherapy drug Taxol alone, another two groups are treated with Taxol in combination with different doses of dammarane sapogenins containing agent. As shown in Fig.10, multidrug resistant cancer cells required as high as 100uM Taxol to kill all cells when applied alone. However, in the presence of dammarane sapogenins, 1/10 of the effective Taxol dose was enough to achieve a 100% kill. In another words, dammarane sapogenins enhanced the efficiency of Taxol 10-fold.

• Dammarane sapogenins versus Gemzar

Fig. 11 shows an example of pancreatic cancer. Gemzar is the first line chemotherapeutic drug for this disease. In this case, pancreatic cancer cells were treated with Gemzar alone and showed 40% cell death (left three columns). In the presence of dammarane sapogenins, 90% of cancer cells were killed using the same dose of Gemzar, demonstrating a significant enhancing effect.

2.3 Dammarane sapogenins can influence the metabolism of P450s and maintain a higher level of anti-cancer drugs

Dammarane sapogenins may potentially increase the effective dose of drugs in our body by influencing the drug metabolizing system in the liver. Most drugs are metabolized by a group of enzymes in the liver called P450s. the activity of P450s determines how rapidly a drug is cleared from our body. While P450s are important to prevent drug induced toxicity, they can also reduce the efficacy of a drug if cleared by the enzymes too soon. Interestingly, dammarane sapogenins have been found to interact with some P450s. In particular, they inhibits the metabolic activity of CYP3A4, 2C9, 2C19, and 2B6, enzymes commonly involved in metabolism of many anti-cancer drugs.
However, the inhibitory effect of dammarane sapogenins also raises a concern when using it together with other drugs. This is because:

• Some anti-cancer drugs rely on P450s to metabolize them into effective compounds. In those cases, dammarane sapogenins cannot be used
• When dammarane sapogenins are used together with anti-cancer drugs, they may increase the side-effects of that drug at its original dose

3. Dammarane sapogenins are potential cancer prevention agents

Prevention is the ultimate approach in the battle against cancer. Dammarane sapogenins can be used as a cancer prevention agent based on the following observations:

1) Dammarane sapogenins arrests the cell cycle in malignant cells and induce differentiation at low concentrations.
2) Dammarane sapogenins inhibit P450s.
3) Dammarane sapogenins inhibit estrogen induced breast cancer growth.

3.1 At low concentration, dammarane sapogenins arrests cancer proliferation and induce differentiation

As shown in fig. 12, cancer cells proliferate through cell division. Cell division occurs in a cycle with 4 major stages from G1 phase to S then G2 and finally reaching the mitosis M phase.

Numerous studies have shown that dammarane sapogenins and derivative compounds can arrest the cell cycle at the G1 phase at low concentrations. In addition, at low concentrations, dammarane sapogenins induce
malignant cell differentiation to the benign form and eventually cause those cells to die with prolonged treatment. Since cancer develops from a single abnormal cell, long term, low dose use of dammarane sapogenins may prevent any potential cancerous cells from growing into a tumor. Actually, in early studies of Korean ginseng, which contain higher levels of dammarane sapogenin-like compounds have also shown effect of tumor prevention both in animal models and epidemiological studies was demonstrated.

Our data demonstrated that dammarane sapogenins and its related compounds can arrest cancer cells to prevent cell division from further progression. Therefore, cell proliferation was stopped. In addition, when cancer is treated with low concentrations of dammarane sapogenins or its derivatives, malignant cells can transform into the benign form, with no cell division and showing differentiation, typical of normal cells. This is demonstrated in fig.13, where a benign cell shape (Fig. 13b) appeared when the malignant melanoma cells (Fig. 13a) were treated for 48 hours. This figure clearly shows untreated malignant cells growing confluently with irregular, spherical shapes while the treated cells became spindle shaped and were loosely distributed (differentiated form).

3.2 Dammarane sapogenins may slightly inhibit P450s and slow down the production of carcinogen.

One of the possible mechanisms for the cancer prevention effect by total ginseng extracts shown in the above studies is slight inhibition of P450s in individuals taking ginseng regularly. P450s are a group of enzymes responsible for metabolizing drugs and all the chemicals in our body. Many environmental pro-carcinogens are metabolized by P450s to intermediates that are highly carcinogenic. For example, one group of P450s is particularly active towards Polycyclic Aromatic Hydrocarbons (PAHs), transforming them into reactive products that covalently bind to DNA, a key event in the initiation of carcinogenesis. Similarly, other groups of P450s activate a variety of substances, such as aromatic amines, amides, some naturally occurring chemicals (e.g. aflatoxin B1) and N-nitrosamines into highly mutagenic and carcinogenic agents. Thus a slight inhibition of P450s would slow down the production of those carcinogens to allow effective removal by the body.

While total ginseng extracts have shown some activity in inhibiting P450s, dammarane sapogenins are the first compounds from ginseng identified with strong inhibitory effects on a number of P450 enzymes. There is clear evidence demonstrating that dammarane sapogenins and dammarane sapogenins containing products can inhibit the metabolic activity of some P450s at relatively low concentrations. Since those P450s have been known for their involvement in turning environmental pro-carcinogens into strong carcinogens, inhibiting those enzymes by dammarane sapogenins suggests a potential use of this compound for cancer prevention.

3.3 Dammarane sapogenins inhibit estrogen induced breast cancer growth.
In addition to the above general cancer prevention activity of dammarane sapogenins, our recent study demonstrated their usefulness in preventing estrogen-dependent cancer in particular. Estrogen is a female hormone with omportant physiological functions. Promotion of breast cancer carcinogenesis by prolonged exposure to estrogens is well-documented. Furthermore, conclusive epidemiological evidence suggests that hormone replacement therapy for healthy postmenopausal women in western countries is associated with a small but substantial increase in breast cancer risk. Our data showed that dammarane sapogenins compete with estrogen for estrogen receptors. Therefore, they prevent estrogen from binding to the receptors for their tumor stimulating effects. In a human breast cancer animal model, dammarane sapogenins or a dammarane sapogenin containing product completely blocked growth of the tumor even in the presence of high levels of estrogen. In addition, when dammarane sapogenins were used in combination with tamoxifen, a significant enhancement of the effect of the latter on killing breast cancer cells occurred (see fig. 15).

Toxicity of dammarane sapogenins is extremely low

Acute toxicity study

In order to propose dammarane sapogenins as an anti-cancer therapeutic, the toxicity most be known. The possible toxic effect of dammarane sapogenins has been extensively studied in animals. Briefly, for the acute toxicity test, rats were orally administered a one time dose of dammarane sapogenins containing agent having 51% of dammarane sapogenins (4,000 mg/kg). The animals appeared quiet and less active with eyes closed for 30 minutes following treatment. The drowsiness disappeared in 4 hours following which the activity, diet and water intake returned to normal. No animal died during 7 days of observation and autopsy did not uncover any abnormalities in the heart, liver, spleen, lung, kidney, stomach or the intestine.

Chronic toxicity study

For the long term toxicity test, animals were orally administered dammarane sapogenins containing agent at high (H) 333.3 mg/kg, medium (M) 131.0 mg/kg and low (L) 51.5 mg/kg dosages respectively. Daily treatment lasted 8 weeks plus a two week post-treatment observation period. Transient hypertrophy in the prostate (187%) and ovary (124%) were found in Group H animals at 24 hours. Similar hypertrophy in the prostate (156%) was found in Group M. However, no hypertrophy was found 2 weeks later. Other minor toxic reactions were only seen in Group H, including drowsiness during week 3 and 4 (back to normal thereafter) and less weight gain (no statistical significance) in the later stage of treatment (week 4-9). Routine blood and organ histological examinations did not show any abnormality in all three groups. Furthermore, blood examination in animals given dammarane sapogenins containing agent have shown no sign of suppression on blood cells (Fig.17,18,19), whereas a common chemotherapy agent Taxol was very toxic to the bone marrow.

Conclusion

Over the past few years, we have examined dammarane sapogenins for its potential anti-cancer activity and have discovered a number of pharmacological activities of this compound.

1. Dammarane sapogenins can arrest cancer cell proliferation and induce their differentiation into a benign form
2. They can also force cancer cells to enter a special cell death pathway named apoptosis, which is a suicidal program usually disabled in cancer cells
3. Dammarane sapogenins can also block the function of a protein in cancer cells termed P-glycoprotein (P-gp) which is responsible for multi-drug resistance. This latter feature allows dammarane sapogenins to dramatically enhance the efficacy of other chemotherapy agents on drug resistant cancer when used in combination.
4. Dammarane sapogenins may interact with P450s to reduce the production of carcinogens in our body, and therefore may decrease the potential risk of getting cancer.
5. Dammarane sapogenins may compete with the estrogen receptor and decrease tumor growth due to stimulation caused by estrogen.

Reference

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