Cell differentiation is a measure of the maturity of a cell. Cells that are fully differentiated (fully mature) resemble their parent cells in form and function, and they proliferate very slowly, if at all. In contrast, immature cells are poorly differentiated, do not yet resemble their parents, and are able to proliferate at a higher rate. Most cancer cells are less differentiated, less mature, than normal cells, allowing cancer cells to proliferate readily. The degree to which a cell differentiates is regulated by gene expression. Therefore, by manipulating gene expression, one can alter the degree of differentiation. A number of natural compounds, discussed below, can induce differentiation in cancer cells, thereby decreasing their proliferation rate and causing them to display fewer malignant characteristics.

Stem Cells

The least differentiated and most prolific cells within the body are called stem cells. In a healthy organism, stem cells act as a source of new cells during tissue repair. Stem cells are capable of both self-renewal (self-replacement) and clonal expansion and so are virtually immortal. Not surprisingly, stem cells are present in high numbers in tissues that constantly renew their population, such as the bone marrow and intestinal lining. Bone marrow cells have a turnover rate of approximately five days, as opposed to several years for some vascular cells. Although stem cells in normal tissues have a high ability to proliferate, their proliferation is tightly regulated, occurring only under specific circumstances.

Small numbers of stem cells are also present in malignant tumors. Unlike stem cell proliferation in normal tissues, that in cancerous tissue is largely unregulated. Furthermore, the daughter cells do not fully differentiate (i.e., acquire the functions of more mature cells), and so the proliferation rate of the offspring remains high. For these reasons, stem cells are the prime targets of cytotoxic chemotherapy and radiotherapy.

Tumors can be described by the degree to which their cells have undergone differentiation; this is referred to as the “grade” of a tumor. Tumors that are poorly differentiated generally grow faster and are assigned a higher grade. The opposite is true for tumors that are well differentiated. If tumor cells do not differentiate at all, the tumor is called anaplastic (literally, not formed). The grading system usually uses a scale of 1 to 3 or 1 to 4, with anaplastic tumors having the highest grade. For example, a well-differentiated tumor may be classified as grade 1, whereas a poorly differentiated one may be grade 4. Most tumors, except perhaps the most anaplastic, contain enough cells that sufficiently differentiate so that a pathologist can determine the tissue of origin. For example, at least a few cells from a bone cancer will differentiate into mature and identifiable bone cells.

Natural Compounds That Induce Differentiation

The cells of most cancers have the potential to differentiate into more mature cells. In other words, many, if not all, cancer cells retain the capacity to express some normal characteristics and, under some circumstances, to suppress malignant behavior.2 Natural compounds and certain drugs can induce differentiation in cancer cells, although some cancers are more easily induced to differentiate than others. The greatest successes so far have been in inducing leukemia cells to differentiate.3 Cells must be in the cell cycle before they will respond to differentiating agents; that is, they must be actively dividing and not in the G0 resting phase .

Leukemia cells are particularly sensitive to differentiating agents in large part because they have a high rate of proliferation relative to cells of other cancers. In contrast to leukemia and other fast-growing cancers, success in inducing the cells of most solid tumors to differentiate has been more sporadic.

We note here that, contrary to popular belief, cancer cells do not generally proliferate at a high rate relative to normal cells. Fast-growing cancers such as leukemias proliferate at roughly the rate of fast-growing normal cells such as bone marrow or hair cells. Fast-growing cancer cells and fast growing normal cells enter the cell cycle about once every two weeks or less, and in some cases once every few days. The cells of other cancers and those of most normal tissues proliferate much more slowly. Often, the rate of a tumor’s growth is measured as its doubling time, the time required for it to double in volume. To provide some examples, the doubling rate of breast cancer is generally about 40 to 100 days, that of lung cancer about 60 to 270 days, of colorectal cancer about 630 days on the average, and that of prostate cancer is commonly greater than 740 days. In general, tumors in younger patients have a faster doubling rate than those in older patients; likewise, tumors arising from metastases tend to have a faster doubling rate than primary tumors. All of these relatively slow-growing cancers are less susceptible to differentiating agents than the faster-growing ones.

We have then the seemingly contradictory result that drugs or other compounds that increase cancer cell proliferation can, when used in combination with differentiating agents, increase cell differentiation and in so doing, ultimately reduce proliferation. As we will later see, some chemotherapy drugs and natural compounds, apart from those that induce differentiation, may also be more effective at inhibiting cancer when cells are actively proliferating; agents that increase proliferation may therefore make these more effective too.

NATURAL COMPOUNDS THAT INDUCE DIFFERENTIATION IN VITRO
Arctigenin
ATRA (vitamin A)
Boswellic acid
Bromelain and other proteolytic enzymes
CAPE
Flavonoids (including apigenin, luteolin, quercetin, genistein,
and daidzein)
Emodin
EPA and DHA
Monoterpenes
Resveratrol
1,25-D3 (vitamin D3)

Not surprisingly, most of the differentiation studies using natural compounds have been conducted on leukemia cells. Still, melanoma, colon, breast, lung, bladder, and brain cancer cells have also been reported to differentiate in some cases.8,_9 Natural compounds that induce differentiation in vitro are listed above.

Of the compounds listed, ATRA (an active metabolite of vitamin A) and 1,25-D3 (the active metabolite of vitamin D3) have received the most research attention. The majority of compounds listed in Table 3.1 induce differentiation within the concentration range of roughly 1 to 50 mM, the exceptions being ATRA and 1,25-D3, which induce differentiation within the concentration range of about 0.01 to 1 mM. This is still above the normal plasma concentrations for these two compounds, however. Some of the compounds listed have also been reported to induce differentiation in vivo. For example, intraperitoneal administration of daidzein (at 25 to 50 mg/kg per day) reduced tumor volume and induced differentiation of leukemia cells held in chambers in mice.10 The equivalent human oral dose is about 1.1 to 2.3 grams per day. The same intraperitoneal dose of boswellic acid also induced differentiation of leukemia cells in mice.11,_12 The equivalent human oral dose is about 340 to 680 milligrams per day. Combinations of ATRA and vitamin D3 at high doses have also been reported to be effective in animals