I have read most of Dr. Murakoshi's thirty-four research articles of which I am aware of him publishing during the past ten years. One was in French and with a couple of others I was limited to only having the article abstracts. His research primarily deals with the structure of cells, especially cancer cells, changes in normal cells that lead to them becoming cancer cells, free radicals and lipid peroxidation, how the immune system fights against cancer and the role of carotenoids in preventing cancers.

I had the pleasure of meeting Dr. Murakoshi in February 1993, and I had the honor of introducing him from the podium during my NNFA lecture on Carotenoids in July 1993. I thought that you might like to know more about his research.

Passwater: Dr. Murakoshi, what led you to this line of research?

Murakoshi: The interesting research of Dr. Hoyoku Nishino, Associate Professor of the Department of Biochemistry at the Kyoto Prefectural University of Medicine, who is the director of our project.

Passwater: Dr. Nishino has published hundreds of scientific articles. What particularly caught your interest?

Murakoshi: His exploration of cancer chemo-prevention by minor dietary constituents such as carotenoids, terpenes, flavones, tannins, curcumin, and so forth.

Passwater: All of those compounds are interesting. I find that carotenoids and bioflavonoids are particularly interesting, and that carotenoids are particularly beautiful. As was pointed out last month, carotenoids are pigments that give us most of the yellows, oranges and red colors of birds, fish, crustaceans, flowers, vegetables and fruits. But there is much more to appreciate about carotenoids when it comes to beauty. For without carotenoids, not only would there be less color in our world, we wouldn't be able to see anything at all. Without carotenoids, there would be no vitamin A and thus no light

gathering pigments in the eye.

Carotenoids and vitamin A both absorb light. The chemical name for vitamin A and its family are "retinol" and "retinoids," respectfully, which express their relationship to the retina of the eye. In the rods, 11-cis retinal combines with opsin to form the red pigment, rhodopsin, which is the primary light sensor in the eye. In the cones, retinal forms the pigment, iodopsin.

Dr. Murakoshi, you, too, seem to favor the carotenoids over the other compounds that Dr. Nishino was investigating. What brought your interest to carotenoids other than beta-carotene?

Murakoshi: When Dr. Nishino and I wished to expand our knowledge of the anti-cancer activity of beta-carotene, it became apparent to us that there was little information available on the anti-cancer activity of the other carotenoids. We were interested in all of the carotenoid constituents of human blood. Since beta-carotene represents less than thirty percent of all of the carotenoids in the blood, we needed to know the effects of the others, which include alpha-carotene, lycopene, and lutein, among others.

I developed the technology to purify alpha-carotene from

the carotenoid mixtures. Palm fruit is especially rich in alpha-carotene as well as beta-carotene, so we compared the anti-cancer activity of alpha-carotene and beta-carotene in vitro and found that alpha-carotene had a stronger anti-cancer activity than beta-carotene in the test system.

Passwater: Other carotenoids have anti-cancer action. At one time, we thought that the anti-cancer action of the carotenoids was related to their ability to make vitamin A in the body, and that vitamin A prevented cancer by maintaining the health of mucous membranes, controlling the maturation of cells, and of course, acting as a weak antioxidant. This reasoning was supported by the fact that vitamin A and beta-carotene both reversed oral pre-cancers called leukoplakia, whereas canthaxanthin, which does not have provitamin A activity does not reverse leukoplakia.

However, beta-carotene and canthaxanthin both inhibit ultraviolet-induced skin cancer, whereas chemically-induced skin cancer was inhibited by beta-carotene, but not by canthaxanthin. This could imply that protection against chemically-induced skin cancer required beta-carotene to be converted into vitamin A, but ultraviolet-induced skin cancer protection did not depend on conversion to vitamin A.

However, epidemiological evidence implies that diets rich in total carotenoids -- not just the carotenoids that possess pro-vitamin A activity -- were very protective against many types of cancers, although high vitamin A intake itself does not appear to be related to decreased risk of cancer.

Moreover, lycopene -- which is not a vitamin A precursor -- was associated with a reduced risk of lung cancer, and canthaxanthin -- which also has no pro-vitamin A activity -- inhibited chemically-induced cancer in animals where vitamin A was not effective.

In cell culture studies, Dr. John Bertram of the University of Hawaii has found that not only alpha-carotene and beta-carotene -- both of which are vitamin A precursors -- but also lutein, lycopene and canthaxanthin -- which are not vitamin A precursors --all inhibit transformation to cancer cells.

Thus, the preponderance of evidence suggests that the antitumor effect of carotenoids is independent of vitamin A. The effects may be due to various actions of the carotenoids in various cancer systems.

Dr. Murakoshi, does your research shed any light on the mechanism of the various carotenoids? Is it their antioxidant action, antiradical action, singlet oxygen quenching, gap-junction communication or what?

Murakoshi: We found that not only beta-carotene, but alpha-carotene, gamma-carotene, lycopene and lutein have anti-cancer activity. We have also investigated the inhibitory effect of various carotenoids on the proliferation of human malignant tumor cells, and found that fucoxanthin, from the brown algae which is common in Japanese food, and halocynthiaxanthin, from sea squirt, and cryptoxanthin have anti-cancer activity on the growth of human neuroblastoma GOTO cells.

Although some carotenoids are more effective than others against certain types of cancer depending on the mechanism of cancer initiation and the molecular structure of the carotenoid, we cannot explain this relationship at this time. The structure does determine how efficient the carotenoid is as an antioxidant, antiradical, singlet oxygen quencher or gap-junction communication enhancer, but more research is required to properly understand just how changes in chemical structure and electron cloud distribution of the carotenoid affects the cancer process.

Table 1 shows how the carotenoids that we tested compare in anti-cancer activity and Table 2 shows the relative singlet oxygen quenching capability of some antioxidants as were determined by Dr. Helmut Sies of the University of Dusseldorf in Germany.

Passwater: In Cancer Research (52:6583-7, 1992) I believe that you found that alpha-carotene was very protective against spontaneous liver cancer and two-stage lung cancer in mice, and more importantly, protective against proliferation of human malignant tumor cells, whereas beta-carotene was not protective (compared to controls). [1] How do you interpret these findings? Is beta-carotene more protective against some cancers and alpha-carotene more effective against others? Or is alpha-carotene always more protective?

Murakoshi: We also believe that beta-carotene is a key cancer preventive agent, since it is abundant in green and yellow vegetables and has the highest pro-vitamin A activity. In fact, previous reports showed both beta-carotene and vitamin A have cancer preventive and anti-cancer activity in-vivo or in-vitro. However, it should also be noted that beta-carotene is often associated with other types of natural carotenoids, such as alpha-carotene, lycopene, lutein, etc. in vegetables and daily food stuffs, and these carotenoids are detectable in human blood and various tissues.

It is of interest that the qualitative patterns -- the carotenoids present -- in the different tissues are generally not similar. We believe that each of the carotenoids has each function -- antioxidant, antiradical, singlet oxygen quencher, gap-junction communication enhancement -- but with varying degrees of strength of each function.

In one case, beta-carotene shows stronger cancer control activity, in another case, alpha-carotene or another carotenoid shows stronger cancer control activity than beta-carotene.

Passwater: In Oncology (49:492-7, 1992) you describe your research showing that mixed carotenoids was protective against intestinal cancer. [2] Did that research involve a different mechanism of cancer promotion?

Murakoshi: In the ENNG-induced mouse duodenal carcinogenesis model, bile acid (glycocholic acid) is considered to be the tumor promoter. Bile acid can cause induction of ornithine decarboxylase (ODC) activity. We found that palm carotenoids have an inhibitory effect on ODC. This ability may be attributed to anti-tumor promoting activity of palm carotenoids. The chief carotenoids of palm are alpha-carotene, beta-carotene and lycopene.

Passwater: In the Journal of the National Cancer Institute, you stated that alpha-carotene was ten times more protective against cancer than beta-carotene. [3] Was that just against proliferation of neuroblasts or for all cancer?

Murakoshi: Alpha-carotene shows the strongest anti-tumor activity on human neuroblastoma GOTO cells. Alpha-carotene also shows stronger anti-proliferative activity than that of beta-carotene on other human malignant cancer cells; pancreatic cancer (PANC-1), glioblastoma (A172), and gastric cancer (HGC-27).

Passwater: What accounts for this difference? Beta-carotene has two beta-ionone rings and thus our bodies, with the help of dioxygenase can split a molecule of beta-carotene into two retinol (vitamin A) molecules. Alpha-carotene is a very similar structure containing the same number of carbon atoms (40), the same nine conjugated double bonds backbone, the same number of ring structures (2), but one ring is a beta-ionone and the other is slightly different in that the double bond is moved one carbon atom. A molecule of alpha-carotene yields only one molecule of vitamin A. Why is alpha-carotene more protective against some cancers than beta-carotene?

Murakoshi: Researchers trying to explain the anti-cancer activity of carotenoids have focused on the carotenoids' properties as singlet oxygen scavengers and antioxidant activities. Dr. P. D. Mascio and his group have reported that the singlet oxygen quenching ability of alpha-carotene is higher than that of beta-carotene. [4] Dr. Lester Packer's group reports that the higher the carotenoid content, the higher the resistance of liver homogenates to in-vitro induced lipid peroxidation, and the values for correlation coefficients for inhibition of lipid peroxidation in the order of alpha-carotene to beta-carotene. [5] Thus, it is possible that the chemopreventive activities of these carotenes remains to be further elucidated.

Passwater: When we eat fruits and vegetables, we get a mixture of carotenoids. In natural foods, do we usually find alpha-carotene along with beta-carotene?

Murakoshi: Yes. Some foods are particularly rich in alpha-carotene. They have plenty of beta-carotene as well. As examples, in carrots, alpha-carotene makes up 33 percent of all the carotenoids, and in pumpkins, alpha-carotene makes up 42 percent of the total carotenes.

Passwater: Would this be a valid reason for selecting a supplement that contains both alpha-carotene and beta-carotene ... and even other carotenoids ... as opposed to taking only beta-carotene?

Murakoshi: Many epidemiological studies have demonstrated that increased dietary intake of both total carotenoids or fruits and vegetables, as well as elevated blood levels of beta-carotene are consistently associated with reduced cancer risk. Since beta-carotene has been considered a key constituent of fruits and vegetables, much of the attention has been focused on beta-carotene. However, beta-carotene is only one of several major dietary carotenoids, and represents less than 30 percent of the total amount of carotenoids circulating in the blood.

In a recent report, Dr. L. L. Marchand and his colleagues suggested that total vegetable intake was more predictive of reduced risk of lung cancer in Hawaii than was the amount of beta-carotene estimated to be in those diets. [6]

Dr. Regina Zeigler's group point out that other carotenoids, other constituents of fruits and vegetables, and dietary patterns closely associated with fruit and vegetable intake need to be explored further as alternatives to the beta-carotene hypothesis. [7] In this context, it is interesting that Dr. N. V. Hicks and colleagues report a protective association of alpha-carotene intake against lung cancer that supports our results. [8] Further investigation of the biological activity, not only of beta-carotene, but also of alpha-carotene and other carotenoids in daily foods, could be important for cancer control.

Passwater: Did you find that a mixture of carotenoids was more effective that either alpha-carotene or beta-carotene alone?

Murakoshi: Palm carotenoids were more effective than either alpha-carotene or beta-carotene alone. This activity may be attributed to either the synergism of the mixture of carotenoids or to the additional activity of the minor carotenoid components such as lycopene and/or gamma-carotene.

Passwater: What is the next step in your research?

Murakoshi: We have enough supporting evidence to justify clinical trials in the chemoprevention of cancer, and that is just what we are going to do.

Passwater: Thank you for sharing your research findings with us, and please let us know how the clinical trials turn out.

1. Potent preventive action of alpha-carotene againstcarcinogenesis: Spontaneous liver carcinogenesis andpromoting stage of lung and skin carcinogenesis in mice aresuppressed more effectively by alpha-carotene than bybeta-carotene.Murakoshi, M.; Nishino, H.; Satomi, Y.; et al.Cancer Res. 52:6583-7 (1992)

2. Palm carotene inhibits tumor-promoting activity of bile acidand intestinal carcinogenesis.Okuzumi, J.; Nishino, H.; Murakoshi, M.; et al.Oncology 49:492-7 (1992)

3. Inhibitory effects of alpha-carotene on proliferation of thehuman neuroblastoma cell line GOTO.Murakoshi, M.; Takayasu, J.; Kimura, O.; et al.J. Natl. Cancer Inst. 81:1649-52 (1989)

4. Lycopene as the most efficient biological carotenoid singletoxygen quencher.Mascio, P. D.; Kaiser, S. and Sies, H.Archiv. Biochem. Biopsy. 274:532-8 (1989)

5. Distribution and antioxidant activity of a palm oil carotenefraction in rats.Serbinova, E.; Choo, M. and Packer, L.Biochem. Int. (1993) (In Press)

6. Vegetable consumption and lung cancer risk:A population-based case-control study in Hawaii.Marchand L. L.; Yoshizawa, C. N.; Kolonel, L. N.; et al.J. Natl. Cancer Inst. 81:1158-64 (1989)

7. Does beta-carotene explain why reduced cancer risk isassociated with vegetable and fruit intake?Ziegler, R. G.; Subar, A. F.; Craft, N. E.; et al.Cancer Res. 52:2060s-2066s (1992)

8. The effect of specific carotenoids on lung cancer risk(Abstract).Hicks, N. V.; Buffler, P. A.; Mackerras, D.; Burau, K. andChristensen, B. L.In: Carotenoids in Human Health, San Diego, The New YorkAcademy of Sciences. (Feb 6-9, 1993)

9. Anti-tumor and anti-tumor promoting activity of alpha- andbeta-carotene.Nishino, H.; Murakoshi, M.; Kitano, H.; et al.Lipid-Soluble Antioxidants; Biochemistry and ClinicalApplications. (1992) pp228-42.