Tungsten-induced carcinogenesis in human bronchial epithelial cells

Abstract

Metals such as arsenic, cadmium, beryllium, and nickel are known human carcinogens; however, other transition metals, such as tungsten (W), remain relatively uninvestigated with regard to their potential carcinogenic activity. Tungsten production for industrial and military applications has almost doubled over the past decade and continues to increase. Here, for the first time, we demonstrate tungsten's ability to induce carcinogenic related endpoints including cell transformation, increased migration, xenograft growth in nude mice, and the activation of multiple cancer-related pathways in transformed clones as determined by RNA sequencing. Human bronchial epithelial cell line (Beas-2B) exposed to tungsten developed carcinogenic properties. In a soft agar assay, tungsten-treated cells formed more colonies than controls and the tungsten-transformed clones formed tumors in nude mice. RNA-sequencing data revealed that the tungsten-transformed clones altered the expression of many cancer-associated genes when compared to control clones. Genes involved in lung cancer, leukemia, and general cancer genes were deregulated by tungsten. Taken together, our data show the carcinogenic potential of tungsten. Further tests are needed, including in vivo and human studies, in order to validate tungsten as a carcinogen to humans.

Keywords: Beas-2B; Cancer; In vitro; Nude mice; RNA-Seq; Tungsten.

Figure 1. Transformation Assay

After 6 weeks of chronic exposure to Na₂WO₄, Beas-2B cells were placed into soft agar. (A) Representative picture of colony formation for each dose showing that W-treated cells formed more transformed colonies than the control. (B) The control cells transformed at an average of 33 colonies per well. At the lowest dose, 50 μM, there was an average of 99 colonies per well. The higher doses, 100 and 250 μM, grew an average of 83 and 89 colonies per well, respectively. W-treated cells show a statistically significantly higher number of transformed colonies than control (p<0.001). *** p-value < 0.001 compared to control

Figure 2. Time Series of Transformation

After three, four, five and six weeks of chronic exposure to sodium tungstate, Beas-2B cells were placed into soft agar in order to evaluate the temporal induction of anchorage independent growth. Untreated controls grew significantly fewer colonies than W-treated cells at each time point. At each of the four time points, colony formation seemed to be variable amongst the doses. This figure shows that after three weeks of chronic treatment, W-treated cells became transformed at each dose. * p-value<0.05, ** p-value<0.01, *** p-value<0.001 compared to control

Figure 3. Scratch Test

500 micron simulated wounds were created in confluent monolayers. (A) Representative pictures are taken in time intervals to capture the migration of the cells showing that the W-transformed clones migrated more quickly than control clone or parental Beas-2B cells. Images were taken at 100X magnification at the same field of view. (B) W-transformed clone cells reduced mean wound width more quickly than either the control clone or parental cells. Wounds were statistically significantly smaller 8 and 20 hours after wound creation in the W-transformed clone as compared to control clone or parental cells (p<0.001). *** p<0.001 compared to control

Figure 4. Tungsten-transformed clones grew tumors in vivo

Twelve, six-week old female athymic nude mice were subcutaneously injected in the left flank with Na₂WO₄ transformed cells or the right flank with sporadic growth control cells. Each mouse is injected on one side with control and the other with tungsten-transformed cells to ensure the same biological environment. This figure shows a representative mouse that has formed tumors on the left side where they were injected with W-transformed cells, unlike the sporadic growth control cells.

Figure 5. Gene expression profiles of W-transformed clones

(A) Heat Map. Hierarchical cluster analysis of significantly differentially expressed genes in W-control clones compared to clones that spontaneously grew in soft agar (control). The bar relates the color code to the expression value of normalized counts with DESeq2 package.