In vitro quantification: Long-term effect of glucose deprivation on various cancer cell lines.

OBJECTIVE: Although metabolic treatment of highly glycolytic cancers and metastases is becoming an important research field, the effects of such treatments are not fully quantified yet. In this article we attempt to quantify the effect of long-term glucose deprivation (similar to ketogenic diets) on cancer cells using in vitro tests. METHODS: Two tumorigenic cell lines were used, namely a metastatic breast and a cervical cancer cell line. The non-tumorigenic control cell line was an immortalized breast cell line. All the cell lines were stabilized at a typical average human blood glucose level of 6 mmol/L. The cell lines were then exposed to the therapeutic blood glucose level of 3 mmol/L for 90 d. RESULTS: The tests indicated that glucose deprivation restricted the different cancer cell lines' growth more than that of non-tumorigenic cells. The different cell lines were also differentially affected, which suggests that long-term glucose deprivation will not be equally effective for different types of cancer. The highly glycolytic breast cancer cell line was most adversely affected, with cell growth decreasing to 30% after 26 d. Cell growth was stable at this level for up to 22 d. Furthermore, all of the other cancer cell lines were similarly affected. CONCLUSIONS: This in vitro data could help to direct future human in vivo tests to find the most therapeutic time (cancer cells at their most vulnerable) for additional short-term adjuvant therapies. Partial recovery of proliferation occurred after 90 d. Therefore, as expected, the results also indicated that without an adjuvant treatment, full extinction cannot be reached with the proposed long-term metabolic treatment. The need for more clinical data on long-term glucose deprivation treatments for cancer is well described in the literature. This paper attempts to add to the available pool of knowledge.

Influence of partial and complete glutamine-and glucose deprivation of breast-and cervical tumorigenic cell lines.

BACKGROUND: Due to their high proliferative requirements, tumorigenic cells possess altered metabolic systems whereby cells utilize higher quantities of glutamine and glucose. These altered metabolic requirements make it of interest to investigate the effects of physiological non-tumorigenic concentrations of glucose and glutamine on tumorigenic cells since deprivation of either results in a canonical amino acid response in mammalian cell. METHODS: The influence of short-term exposure of tumorigenic cells to correlating decreasing glutamine- and glucose quantities were demonstrated in a highly glycolytic metastatic breast cell line and a cervical carcinoma cell line. Thereafter, cells were propagated in medium containing typical physiological concentrations of 1 mM glutamine and 6 mM glucose for 7 days. The effects on morphology were investigated by means of polarization-optical transmitted light differential interference contrast. Flow cytometry was used to demonstrate the effects of glutamine-and glucose starvation on cell cycle progression and apoptosis induction. Fluorometrics were also conducted to investigate the effects on intrinsic apoptosis induction (mitocapture), reactive oxygen species production (2,7-dichlorofluorescein diacetate) and acidic vesicle formation (acridine orange). RESULTS: Morphological data suggests that glutamine-and glucose deprivation resulted in reduced cell density and rounded cells. Glutamine-and glucose starvation also resulted in an increase in the G2M phase and a sub-G1 peak. Complete starvation of glutamine and glucose resulted in the reduction of the mitochondrial membrane potential in both cell lines with MDA-MB-231 cells more prominently affected when compared to HeLa cells. Further, starved cells could not be rescued sufficiently by propagating since cells possessed an increase in reactive oxygen species, acidic compartments and vacuole formation. CONCLUSION: Starvation from glutamine and glucose for short periods resulted in decreased cell density, rounded cells and apoptosis induction by means of reactive oxygen species generation and mitochondrial dysfunction. In addition, the metastatic cell line reacted more prominently to glutamine-and glucose starvation due to their highly glycolytic nature. Satisfactory cellular rescue was not possible as cells demonstrated oxidative stress and depolarized mitochondrial membrane potential. This study contributes to the knowledge regarding the in vitro effects and signal transduction of glucose and/or l-glutamine deprivation in tumorigenic cell lines.

Tumor cell culture survival following glucose and glutamine deprivation at typical physiological concentrations.

OBJECTIVE: Most glucose (and glutamine)-deprivation studies of cancer cell cultures focus on total depletion, and are conducted over at least 24 h. It is difficult to extrapolate findings from such experiments to practical anti-glycolytic treatments, such as with insulin-inhibiting diets (with 10%-50% carbohydrate dietary restriction) or with isolated limb perfusion therapy (which usually lasts about 90 min). The aim of this study was to obtain experimental data on the effect of partial deprivation of d-glucose and l-glutamine (to typical physiological concentrations) during 0 to 6-h exposures of HeLa cells. METHODS: HeLa cells were treated for 0 to 6 h with 6 mM d-glucose and 1 mM l-glutamine (normal in vivo conditions), 3 mM d-glucose and 0.5 mM l-glutamine (severe hypoglycemic conditions), and 0 mM d-glucose and 0 mM l-glutamine ("starvation"). Polarization-optical differential interference contrast and phase-contrast light microscopy were employed to investigate morphologic changes. RESULTS: Reduction of glucose levels from 6 to 3 mM (and glutamine levels from 1 to 0.5 mM) brings about cancer cell survival of 73% after 2-h exposure and 63% after 4-h exposure. Reducing glucose levels from 6 to 0 mM (and glutamine levels from 1 to 0 mM) for 4 h resulted in 53% cell survival. CONCLUSION: These data reveal that glucose (and glutamine) deprivation to typical physiological concentrations result in significant cancer cell killing after as little as 2 h. This supports the possibility of combining anti-glycolytic treatment, such as a carbohydrate-restricted diet, with chemotherapeutics for enhanced cancer cell killing.

A practical quantification of blood glucose production due to high-level chronic stress.

Blood glucose (BG) is the primary metabolic fuel for, among others, cancer cell progression, cardiovascular disease and inflammation. Stress is an important contributor to the amount of BG produced especially by the liver. In this paper, we attempt to quantify the BG production due to chronic (in the order of weeks) high-level psychological stress in a manner that a lay person will understand. Three independent approaches were used. The first approach was based on a literature survey of stress hormone data from healthy individuals and its subsequent mathematical manipulation. The next approach was a deductive process where BG levels could be deduced from published stress data of large cardiovascular clinical trials. The third approach used empirical BG data and a BG simulation model. The three different methods produced an average BG increase of 2.2-fold above basal for high levels of stress over a period of more than a day. The standard deviation normalized to the average value was 4.5%.

High-glycolytic cancers and their interplay with the body's glucose demand and supply cycle.

Many difficult-to-treat solid cancer tumours and metastases have high-glucose uptake, usually under hypoxic conditions. Hypoxic tumours suppress the immune system and are insensitive to traditional chemoradiotherapies. The only therapy usually available is surgical resection. However, with widespread metastases, surgery often becomes unviable. Surgery in itself can also result in metastasis. The need for investigating adjuvant treatments is obvious. Here we investigate whether the high-glucose uptake of hypoxic tumours could lead to such a treatment. Before any treatment can be hypothesised, it is crucial to understand how this glycolytic cancer phenotype fits in with the normal body's blood glucose cycle. The brain creates the healthy body's largest demand for blood glucose (BG) and ensures a very high level of control on in vivo supply. It is hypothesised that, through somatic evolution, high-glycolytic cancer cells opportunistically tap into this very stable energy environment. It is shown that therapies which target the glycolytic cancers' high BG needs cannot be developed without addressing the brain's energy needs. Based on this knowledge, and to initiate thinking on potential BG therapies, a first attempt is made at hypotheses for potential control of the in vivo brain demand as well as the available in vivo BG. The aim is to adversely affect primary as well as metastatic tumours without damaging brain and innocent bystander cells.